Methods for the early diagnosis of ovarian cancer

ABSTRACT

The present invention discloses the protease stratum corneum chymotrytic enzyme (SCCE) is specifically over-expressed in ovarian and other malignancies. A number of SCCE peptides can induce immune responses to SCCE, thereby demonstrating the potential of these peptides in monitoring and the development of immunotherapies for ovarian and other malignancies.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part application which claims thebenefit of priority under 35 USC §120 of U.S. Ser. No. 10/372,521, filedFeb. 21, 2003, which is a continuation-in-part application of U.S. Ser.No. 09/918,243, filed Jul. 30, 2001, which is a continuation-in-partapplication of U.S. Ser. No. 09/905,083, filed Jul. 13, 2001, which is adivisional application of U.S. Pat. No. 6,294,344, which is acontinuation-in-part application of U.S. Pat. No. 6,303,318, whichclaims benefit of provisional patent application U.S. Ser. No.60/041,404, filed Mar. 19, 1997, now abandoned.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] Generally, the present invention relates to the fields ofmolecular biology and medicine. More specifically, the present inventionis in the field of cancer research, especially ovarian cancer diagnosis.

[0004] 2. Background of the Invention

[0005] In order for malignant cells to grow, spread or metastasize, theymust have the capacity to invade local host tissue, dissociate or shedfrom the primary tumor, enter and survive in the bloodstream, implant byinvasion into the surface of the target organ and establish anenvironment conducive for new colony growth (including the induction ofangiogenic and growth factors). During this progression, natural tissuebarriers such as basement membranes and connective tissue have to bedegraded. These barriers include collagen, laminin, fibronectin,proteoglycans and extracellular matrix glycoproteins. Degradation ofthese natural barriers, both those surrounding the primary tumor and atthe sites of metastatic invasion, is believed to be brought about by theaction of a matrix of extracellular proteases.

[0006] Proteases have been classified into four families: serineproteases, metallo-proteases, aspartic proteases and cysteine proteases.Many proteases have been shown to be involved in human disease processesand these enzymes are targets for the development of inhibitors as newtherapeutic agents. Certain individual proteases are induced andoverexpressed in a diverse group of cancers, and as such, are potentialcandidates for markers of early diagnosis and targets for possibletherapeutic intervention. A group of examples are shown in Table 1.TABLE 1 Known proteases expressed in various cancers Gastric BrainBreast Ovarian Serine uPA uPA NES-1 NES-1 Proteases: PAI-1 PAI-1 uPA uPAtPA PAI-2 Cysteine CatSCCE B CatSCCE L CatSCCE B CatSCCE B Proteases:CatSCCE L CatSCCE L CatSCCE L Metallo- Matrilysin* MatrilysinStromelysin-3 MMP-2 proteases: Collagenase* Stromelysin MMP-8Stromelysin-1* Gelatinase B MMP-9 Gelatinase A

[0007] There is a good body of evidence supporting the downregulation orinhibition of individual proteases and the reduction in invasivecapacity or malignancy. In work by Clark et al., inhibition of in vitrogrowth of human small cell lung cancer was demonstrated using a generalserine protease inhibitor. More recently, Torres-Rosedo et al. (1993)demonstrated an inhibition of hepatoma tumor cell growth using specificantisense inhibitors for the serine protease hepsin. Metastaticpotential of melanoma cells has also been shown to be reduced in a mousemodel using a synthetic inhibitor (batimastat) of metallo-proteases.Powell et al. (1993) presented evidence to confirm that the expressionof extracellular proteases in a non-metastatic prostate cancer cell lineenhances their malignant progression. Specifically, enhanced metastasiswas demonstrated after introducing and expressing the PUMP-1metallo-protease gene. There is also a body of data to support thenotion that expression of cell surface proteases on relativelynon-metastatic cell types increases the invasive potential of suchcells.

[0008] To date, ovarian cancer remains the number one killer of womenwith gynecologic malignant hyperplasia. Approximately 75% of womendiagnosed with such cancers are already at an advanced stage (III andIV) of the disease at their initial diagnosis. During the past 20 years,neither diagnosis nor five-year survival rates have greatly improved forthese patients. This is substantially due to the high percentage ofhigh-stage initial detection of the disease. Therefore, the challengeremains to develop new markers that improve early diagnosis and therebyreduce the percentage of high-stage initial diagnoses. The ability todisengage from one tissue and re-engage the surface of another tissue iswhat provides for the morbidity and mortality associated with thisdisease. Therefore, extracellular proteases may be good candidates formarkers of malignant ovarian hyperplasia.

[0009] Thus, the prior art is deficient in a tumor marker useful as anindicator of early disease, particularly for ovarian cancers. Thepresent invention fulfills this long-standing need and desire in theart.

SUMMARY OF THE INVENTION

[0010] This invention allows for the detection of cancer, especiallyovarian cancer, by screening for stratum corneum chymotrytic enzyme(SCCE) mRNA in tissue. Stratum corneum chymotrytic enzyme specificallyassociates with the surface of 80 percent of ovarian and other tumors.Proteases are considered to be an integral part of tumor growth andmetastasis, and therefore, markers indicative of their presence orabsence are useful for the diagnosis of cancer. Furthermore, the presentinvention is useful for treatment (i.e., by inhibiting SCCE orexpression of SCCE), for targeted therapy, for vaccination, etc.

[0011] The present invention provides methods of vaccinating anindividual against SCCE or produce immune-activated cells directedtoward SCCE by inoculating an individual with an expression vectorencoding a SCCE protein or a fragment thereof.

[0012] The present invention also provides methods of immunotherapytargeted toward SCCE in an individual, involving the steps of generatingdendritic cells in vitro from peripheral blood drawn from an individual,loading these dendritic cells with SCCE protein or a fragment thereof,then transferring these dendritic cells back to the individual in singleor multiple doses. SCCE-loaded or SCCE-expressing dendritic cells canalso be used to stimulate SCCE-specific T cell responses in vitro,followed by adoptive immunotherapy in which the individual is givenautologous SCCE-specific T cells.

[0013] There is also provided a method of monitoring the efficacy ofvaccinating an individual with SCCE or SCCE peptide. The methodcomprises measuring immune responses in response to said SCCE or SCCEpeptide, wherein induction of immune responses to said SCCE or SCCEpeptide indicates that said individual has been vaccinated against SCCE.

[0014] In another embodiment of the present invention, there areprovided methods of inhibiting expression of SCCE in a cell byintroducing into a cell a vector encoding an antisense SCCE RNA or anantibody that binds the SCCE protein.

[0015] In yet another embodiment of the present invention, there isprovided a method of targeted therapy to an individual using a compoundthat has a targeting moiety specific for SCCE and a therapeutic moiety.

[0016] In still yet another embodiment of the present invention, thereare provided compositions comprising immunogenic fragments of SCCEprotein or an oligonucleotide having a sequence complementary to SEQ IDNo.30. Also embodied is a method of treating a neoplastic state in anindividual with an effective dose of the above-describedoligonucleotide.

[0017] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows agarose gel comparison of PCR products derived fromnormal and carcinoma cDNA.

[0019]FIG. 2 shows Northern blot analysis of ovarian tumors using SCCE,SCCE, PUMP-1, TADG-14 and β-tubulin probes.

[0020]FIG. 3 shows amplification with serine protease redundant primers:

[0021] histidine sense (S1) with aspartic acid antisense (AS 1), usingnormal cDNA (Lane 1) and tumor cDNA (Lane 2); and histidine sense (S1)with serine antisense (AS2), using normal cDNA (Lane 3) and tumor cDNA(Lane 4).

[0022]FIG. 4 shows amplification with cysteine protease redundantprimers. Normal (Lane 1), low malignant potential (Lane 2), seriouscarcinoma (Lane 3), mucinous carcinoma (Lane 4), and clear cellcarcinoma (Lane 5).

[0023]FIG. 5 shows amplification with metallo-protease redundantprimers. Normal (Lane 1), low malignant potential (Lane 2), seriouscarcinoma (Lane 3), mucinous carcinoma (Lane 4), and clear cellcarcinoma (Lane 5).

[0024]FIG. 6 shows quantitative PCR analysis of SCCE expression. Cases3, 4 and 9 are normal ovaries. Cases 19, 21, 14, 15 and 16 are LMPtumors. Cases 43, 23, 36 and 37 are ovarian carcinomas. Expresssionlevels of stratum corneum chymotrytic enzyme relative to β-tubulin aresignificantly elevated in tumor Cases 19, 14, 15, 16, 43, 23, 36 and 37compared to that of normal ovaries.

[0025]FIG. 7A shows Northern blot analysis of stratum corneumchymotrytic enzyme mRNA from normal ovary and ovarian carcinomas. Lane1, normal ovary (case 10); Lane 2, serous carcinoma (case 35); Lane 3,mucinous carcinoma (case 48); Lane 4, endometrioid carcinoma (case 51);and Lane 5, clear cell carcinoma (case 54). Two transcripts (1.2 and 2.0kb) were detected in all of the subtypes of carcinoma (lanes 2-5).

[0026]FIGS. 7B and 7C show that normal human adult tissues (spleen,thymus, prostate, testis, ovary, small intestine, colon, peripheralblood leukocyte, heart, brain, placenta, lung, liver, skeletal muscle,kidney and pancreas) and normal human fetal tissues (brain, lung, liverand kidney) examined showed no visible SCCE transcripts.

[0027]FIG. 8 shows the ratio of SCCE expression to expression ofβ-tubulin in normal ovary, LMP tumor and ovarian carcinoma. SCCE mRNAexpression levels were significantly elevated in LMP tumor (p<0.05) andcarcinoma (p<0.00 1) compared to that in normal ovary. All 10 cases ofnormal ovaries showed a low level of SCCE mRNA expression.

[0028]FIG. 9 shows MDA-MB-435S (Lanes 1 & 3) and HeLa (Lanes 2 & 4) celllysates were separated by SDS-PAGE and immunoblotted. Lanes 1 & 2 wereprobed with rabbit pre-immune serum as a negative control. Lanes 3 & 4were probed with polyclonal rabbit antibodies generated to peptidesderived from SCCE protein sequence.

[0029]FIG. 10A shows normal surface ovarian epithelium. Little SCCEexpression was observed (normal ovary, X100). FIG. 10B is a negativecontrol section for FIG. 10A. No nonspecific staining was observed(Normal ovary, X100). FIG. 10C shows positive SCCE staining localized inthe cytoplasm and the cell membrane of ovarian cancer cells (case 947,clear cell adenocarcinoma, X100). FIG. 10D is a negative control sectionfor FIG. 10C. No nonspecific staining was observed (case 947, clear celladenocarcinoma, X100). FIG. 10E is positive stratum corneum chymotryticenzyme staining localized in the cytoplasm and the cell membrane ofovarian cancer cells. Mucin in the glands also showed positive stratumcorneum chymotrytic enzyme staining (case 947, clear celladenocarcinoma, X100). FIG. 10F is a negative control section for FIG.10E. No nonspecific staining was observed (case 947, clear celladenocarcinoma, X100).

[0030]FIG. 11A shows Northern blot analysis of hepsin expression innormal ovary and ovarian carcinomas. Lane 1, normal ovary (case 10);lane 2, serous carcinoma (case 35); lane 3, mucinous carcinoma (case48); lane 4, endometrioid carcinoma (case 51); and lane 5, clear cellcarcinoma (case 54). In cases 35, 51 and 54, more than a 10-foldincrease in the hepsin 1.8 kb transcript abundance was observed.Northern blot analysis of hepsin in normal human fetal (FIG. 11B) andadult tissues (FIG. 11C). Significant overexpression of the hepsintranscript is noted in both fetal liver and fetal kidney. Notably,hepsin overexpression is not observed in normal adult tissue. Slightexpression above the background level is observed in the adult prostate.

[0031]FIG. 12A shows hepsin expression in normal (N), mucinous (M) andserous (S) low malignant potential (LMP) tumors and carcinomas (CA).FIG. 12B shows a bar graph of expression of hepsin in 10 normal ovariesand 44 ovarian carcinoma samples.

[0032]FIG. 13 shows a comparison by quantitative PCR of normal andovarian carcinoma expression of mRNA for protease M.

[0033]FIG. 14 shows the TADG-12 catalytic domain including an insertnear the His 5′-end.

[0034]FIG. 15A shows northern blot analysis comparing TADG- 14expression in normal and ovarian carcinoma tissues. FIG. 15B showspreliminary quantitative PCR amplification of normal and carcinoma cDNAsusing specific primers for TADG-14.

[0035]FIG. 16A shows northern blot analysis of the PUMP-I gene in normalovary and ovarian carcinomas. FIG. 16B shows northern blot analysis ofthe PUMP-1 gene in human fetal tissue. FIG. 16C shows northern blotanalysis of the PUMP-1 gene in adult tissues.

[0036]FIG. 17A shows a comparison of PUMP-1 expression in normal andcarcinoma tissues using quantitative PCR with an internal β-tubulincontrol. FIG. 17B shows the ratio of mRNA expression of PUMP-1 comparedto the internal control β-tubulin in 10 normal and 44 ovariancarcinomas.

[0037]FIG. 18 shows a comparison of Cathepsin L expression in normal andcarcinoma tissues using quantitative PCR with an internal β-tubulincontrol.

[0038]FIG. 19 is a summary of PCR amplified products for the hepsin,SCCE, protease M, PUMP-1 and Cathepsin L genes.

[0039]FIG. 20 shows CD8⁺ CTL recognition of SCCE 5-13 peptide in a 5 hr⁵¹Cr release assay. Targets were LCL loaded with SCCE 5-13 () andcontrol LCL (∘).

[0040]FIG. 21 shows CD8⁺ CTL recognition of SCCE 123-131 peptide in a 5hr ⁵¹Cr release assay. Targets were LCL loaded with SCCE 123-131 () andcontrol LCL (∘).

[0041]FIG. 22 shows peptide-specific CD8⁺ CTL recognition ofendogenously processed and presented hepsin tumor antigen. CTL werederived by stimulation with dendritic cells pulsed with SCCE peptide123-131. Cytotoxicity was tested in a standard hours ⁵¹Cr-release assayagainst autologous macrophages infected with Ad-GFP/SCCE (♦),macrophages infected with Ad-GFP-hepsin (▴), macrophages pulsed withSCCE 123-131 peptide (▪), or control untreated macrophages ().

[0042]FIG. 23 shows CD8⁺ CTL specific for SCCE 123-131 lyse ovariantumor cells. Target cells are autologous LCL loaded with 5 ug/ml peptide(♦), control LCL (open diamond), CaOV-3 ovarian tumor cells (♦), CaOV-3tumor cells plus 1/25 dilution of BB7.2 ascites (▪), CaOV-3 tumor cellsplus 50 ug/ml W6/32 (), and K562 cells (∘).

[0043]FIG. 24 shows SCCE 110-139-specific CD4⁺ T cells from donor 1(DR1, DR7 haplotype) lysed autologous peptide-pulsed LCL. Target cellswere autologous LCL pulsed over night with 50 mg/ml peptide (▪) orcontrol LCL (♦).

[0044]FIG. 25 shows SCCE 110-139-specific CD4⁺ T cells from donor 2(DR3, DR6 haplotype) lysed autologous peptide-loaded dendritic cells.Target cells were immature dendritic cells loaded with 50 mg/ml peptideand then matured for 48 hours (▪) or control mature dendritic cells (♦).

[0045]FIG. 26 shows SCCE 110-139-specific CD8⁺ CTL responses. LCL cellswere pulsed over night with 50 mg/ml SCCE 110-139. Target cells werepeptide-pulsed autologous LCL (♦), peptide-pulsed HLA A2.1-matchedallogeneic LCL (▪), or peptide-pulsed HLA Class I-mismatched allogeneicLCL (). Control unpulsed LCL cells were not recognized.

[0046]FIG. 27 shows CD8⁺ T cells specific for SCCE peptide 123-131recognized and lysed dendritic cells loaded with SCCE peptide 110-139.Immature dendritic cells were loaded with 50 mg/ml peptide and thenmatured for 48 hours before use as target cells. Target cells werepeptide 110-139-loaded dendritic cells (▪) or control dendritic cells(♦).

DETAILED DESCRIPTION OF THE INVENTION

[0047] This invention identifies stratum corneum chymotrytic enzyme(SCCE) as a marker for ovarian tumor cells. In various combinations withother proteases, stratum corneum chymotrytic enzyme expression ischaracteristic of individual tumor types. Such information can providethe basis for diagnostic tests (assays or immunohistochemistry) andprognostic evaluation (depending on the display pattern).

[0048] Long-term treatment of tumor growth, invasion and metastasis hasnot succeeded with existing chemotherapeutic agents. Most tumors becomeresistant to drugs after multiple cycles of chemotherapy. The presentinvention identifies SCCE as a new therapeutic intervention targetutilizing either antibodies directed at the protease, antisense vehiclesfor downregulation or protease inhibitors both from establishedinhibition data and/or for the design of new drugs.

[0049] The present invention provides a method of vaccinating anindividual against SCCE, comprising the steps of inoculating anindividual with an expression vector encoding a SCCE peptide or withpeptide-loaded dendritic cells. Expression of the SCCE peptide elicitsan immune response in the individual, thereby vaccinating the individualagainst SCCE. Generally, this method is applicable when the individualhas cancer or is at risk of getting a cancer such as ovarian cancer,lung cancer, prostate cancer and colon cancer. Sequences of preferredSCCE peptides are shown in SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86,99 and 137.

[0050] The present invention also provides a method of producingimmune-activated cells directed toward SCCE, comprising the steps ofexposing immune cells to SCCE protein or fragment thereof. Typically,exposure to SCCE protein or fragment thereof activates the immune cells,thereby producing immune-activated cells directed toward SCCE.Generally, the immune-activated cells are B-cells, T-cells and/ordendritic cells. Preferably, the SCCE fragment is a 9-residue fragmentup to a 30-residue fragment, and more preferably, the fragment is SEQ IDNos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and 137. Oftentimes, thedendritic cells are isolated from an individual prior to exposure andthen reintroduced into the individual subsequent to the exposure.Typically, the individual has cancer or is at risk of getting a cancersuch as ovarian cancer, lung cancer, prostate cancer and colon cancer.

[0051] The present invention also provides methods of immunotherapytargeted toward SCCE in an individual. In one embodiment, the methodinvolves generating dendritic cells in vitro from peripheral blood drawnfrom the individual, loading these dendritic cells with SCCE protein ora fragment thereof by lipofection or other means, then transferringthese dendritic cells back to the individual in single or multipledoses. SCCE may also be expressed in these dendritic cells followingtransduction with a recombinant DNA vector. Alternatively, SCCE-loadedor SCCE-expressing dendritic cells can be used to stimulatehepsin-specific T cell responses in vitro, followed by adoptiveimmunotherapy in which the individual is given autologous SCCE-specificT cells. Typically, the individual has cancer or is at risk of getting acancer such as ovarian cancer, lung cancer, prostate cancer and coloncancer. In general, a full length or a fragment of SCCE protein isexpressed in the isolated dendritic cells. Preferably, the fragment is a9-residue fragment up to a 30-residue fragment, and more preferably, thefragment is SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and 137.

[0052] There is also provided a method of monitoring the efficacy ofvaccinating an individual with SCCE or SCCE peptide such as those shownin SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and 137. The methodcomprises isolating T cells or CD8⁺ T cells from the vaccinatedindividual and measuring immune responses specific to said SCCE or SCCEpeptide. An increased level of immune responses compared to thoseexhibited by cells from normal individual indicates that said individualhas been vaccinated by said SCCE or SCCE peptide. In general, theindividual is vaccinated to SCCE if there is an increased level ofSCCE-specific T cells proliferation, an increased frequency ofSCCE-specific T cells or an increased frequency of SCCE-specificcytokine-secreting T cells. Standard assays well-known in the art suchas tetramer analysis and ELISPOT assay can be used to determine thefrequency of SCCE-specific T cells and the frequency of SCCE-specificcytokine-secreting T cells respectively.

[0053] In another aspect of the present invention, there is provided amethod of inhibiting expression of SCCE in a cell, comprising the stepof introducing into a cell a vector comprises a sequence complementaryto SEQ ID No.30, wherein expression of the vector produces SCCEantisense RNA in the cell. The SCCE antisense RNA hybridizes toendogenous SCCE mRNA, thereby inhibiting expression of SCCE in the cell.

[0054] Expression of SCCE can also be inhibited by antibody. An antibodyspecific for a SCCE protein or a fragment thereof is introduced into acell, and binding of the antibody to SCCE would inhibit the SCCEprotein. Preferably, the SCCE fragment is a 9-residue fragment up to a30-residue fragment, and more preferably, the fragment is SEQ ID Nos.31,32,33,34, 35,36, 80, 86, 99 and 137.

[0055] The present invention is also directed toward a method oftargeted therapy to an individual, comprising the step of administeringa compound to an individual, wherein the compound has a targeting moietyspecific for SCCE and a therapeutic moiety. Preferably, the targetingmoiety is an antibody specific for SCCE, a ligand or ligand bindingdomain that binds SCCE. Likewise, the therapeutic moiety is preferably aradioisotope, a toxin, a chemotherapeutic agent, an immune stimulant orcytotoxic agent. Generally, the individual suffers from a disease suchas ovarian cancer, lung cancer, prostate cancer, colon cancer or anothercancer in which hepsin is overexpressed.

[0056] The present invention is further directed toward an immunogeniccomposition, comprising an appropriate adjuvant and an immunogenic fulllength SCCE protein or a fragment thereof. Preferably, the fragment is a9-residue fragment up to a 30-residue fragment, and more preferably, thefragment is SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and 137.

[0057] The present invention also provides an oligonucleotide having asequence complementary to SEQ ID No.30 or a fragment thereof. Thepresent invention further provides a composition comprising theabove-described oligonucleotide and a physiologically acceptablecarrier, and a method of treating a neoplastic state in an individual,comprising the step of administering to the individual an effective doseof the above-described oligonucleotide. Typically, the neoplastic statemay be ovarian cancer, breast cancer, lung cancer, colon cancer,prostate cancer or another cancer in which SCCE is overexpressed.

[0058] It will be apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

[0059] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J.

[0060] Higgins eds. 1985); “Transcription and Translation” (B. D. Hames& S. J. Higgins eds. 1984); “Animal Cell Culture” (R. I. Freshney, ed.1986); “Immobilized Cells And Enzymes” (IRL Press, 1986); B. Perbal, “APractical Guide To Molecular Cloning” (1984). Therefore, if appearingherein, the following terms shall have the definitions set out below.

[0061] As used herein, the term “cDNA” shall refer to the DNA copy ofthe mRNA transcript of a gene.

[0062] As used herein, the term “PCR” refers to the polymerase chainreaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202to Mullis, as well as other improvements now known in the art.

[0063] The present invention comprises a vector comprising a DNAsequence which encodes a SCCE protein, wherein said vector is capable ofreplication in a host, and comprises, in operable linkage: a) an originof replication; b) a promoter; and c) a DNA sequence coding for saidSCCE protein. Preferably, the vector of the present invention contains aportion of the DNA sequence shown in SEQ ID No. 30. Vectors may be usedto amplify and/or express nucleic acid encoding a SCCE protein, afragment of SCCE protein, or an antisense SCCE mRNA.

[0064] An expression vector is a replicable construct in which a nucleicacid sequence encoding a polypeptide is operably linked to suitablecontrol sequences capable of effecting expression of the polypeptide ina cell. The need for such control sequences will vary depending upon thecell selected and the transformation method chosen.

[0065] Generally, control sequences include a transcriptional promoterand/or enhancer, suitable mRNA ribosomal binding sites and sequenceswhich control the termination of transcription and translation. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing appropriate transcriptional andtranslational control signals. See, for example, techniques described inSambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd Ed.),Cold Spring Harbor Press, N.Y. A gene and its transcription controlsequences are defined as being “operably linked” if the transcriptioncontrol sequences effectively control transcription of the gene. Vectorsof the invention include, but are not limited to, plasmid vectors andviral vectors. Preferred viral vectors of the invention are thosederived from retroviruses, adenovirus, adeno-associated virus, SV40virus, or herpes viruses.

[0066] As used herein, the term “host” is meant to include not onlyprokaryotes but also eukaryotes such as yeast, plant and animal cells. Arecombinant DNA molecule or gene which encodes a human SCCE protein ofthe present invention can be used to transform a host using any of thetechniques commonly known to those of ordinary skill in the art.Especially preferred is the use of a vector containing coding sequencesfor the gene which encodes a human SCCE protein of the present inventionfor purposes of prokaryote transformation. Prokaryotic hosts may includeE. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis.Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cellsand insect cells.

[0067] The term “oligonucleotide”, as used herein, is defined as amolecule comprised of two or more ribonucleotides, preferably more thanthree. Its exact size will depend upon many factors, which, in turn,depend upon the ultimate function and use of the oligonucleotide. Theterm “primer”, as used herein, refers to an oligonucleotide, whetheroccurring naturally (as in a purified restriction digest) or producedsynthetically, and which is capable of initiating synthesis of a strandcomplementary to a nucleic acid when placed under appropriateconditions, i.e., in the presence of nucleotides and an inducing agent,such as a DNA polymerase, and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, sequenceand/or homology of primer and the method used. For example, indiagnostic applications, the oligonucleotide primer typically contains15-25 or more nucleotides, depending upon the complexity of the targetsequence, although it may contain fewer nucleotides.

[0068] The primers herein are selected to be “substantially”complementary to particular target DNA sequences. This means that theprimers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment (i.e., containing a restriction site) may beattached to the 5′ end of the primer, with the remainder of the primersequence being complementary to the strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementarywith the sequence to hybridize therewith and form the template forsynthesis of the extension product.

[0069] As used herein, “substantially pure DNA” means DNA that is notpart of a milieu in which the DNA naturally occurs, by virtue ofseparation (partial or total purification) of some or all of themolecules of that milieu, or by virtue of alteration of sequences thatflank the claimed DNA. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote; or which exists as a separate molecule (e.g., acDNA or a genomic or cDNA fragment produced by polymerase chain reaction(PCR) or restriction endonuclease digestion) independent of othersequences. It also includes a recombinant DNA which is part of a hybridgene encoding additional polypeptide sequence, e.g., a fusion protein.Also included is a recombinant DNA which includes a portion of thenucleotides listed in SEQ ID No. 30 and which encodes an alternativesplice variant of SCCE.

[0070] The DNA may have at least about 70% sequence identity to thecoding sequence of the nucleotides listed in SEQ ID No. 30, preferablyat least 75% (e.g., at least 80%); and most preferably at least 90%. Theidentity between two sequences is a direct function of the number ofmatching or identical positions. When a position in both of the twosequences is occupied by the same monomeric subunit, e.g., if a givenposition is occupied by an adenine in each of two DNA molecules, thenthey are identical at that position. For example, if 7 positions in asequence 10 nucleotides in length are identical to the correspondingpositions in a second 10-nucleotide sequence, then the two sequenceshave 70% sequence identity. The length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 100 nucleotides. Sequence identity is typically measuredusing sequence analysis software (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group (GCG), University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

[0071] Further included in this invention are SCCE proteins which areencoded, at least in part, by portions of SEQ ID No. 30, e.g., productsof alternative mRNA splicing or alternative protein processing events,or in which a section of SCCE sequence has been deleted. The fragment,or the intact SCCE polypeptide, may be covalently linked to anotherpolypeptide, e.g., one which acts as a label, a ligand or a means toincrease antigenicity.

[0072] A substantially pure SCCE protein may be obtained, for example,by extraction from a natural source; by expression of a recombinantnucleic acid encoding a SCCE polypeptide; or by chemically synthesizingthe protein. Purity can be measured by any appropriate method, e.g.,column chromatography, such as immunoaffinity chromatography using anantibody specific for SCCE, polyacrylamide gel electrophoresis, or HPLCanalysis. A protein is substantially free of naturally associatedcomponents when it is separated from at least some of those contaminantswhich accompany it in its natural state. Thus, a protein which ischemically synthesized or produced in a cellular system different fromthe cell from which it naturally originates will be, by definition,substantially free from its naturally associated components.Accordingly, substantially pure proteins include eukaryotic proteinssynthesized in E. coli, other prokaryotes, or any other organism inwhich they do not naturally occur.

[0073] In addition to substantially full-length proteins, the inventionalso includes fragments (e.g., antigenic fragments) of the SCCE protein.As used herein, “fragment,” as applied to a polypeptide, will ordinarilybe at least 10 residues, more typically at least 20 residues, andpreferably at least 30 (e.g., 50) residues in length, but less than theentire, intact sequence. Fragments of the SCCE protein can be generatedby methods known to those skilled in the art, e.g., by enzymaticdigestion of naturally occurring or recombinant SCCE protein, byrecombinant DNA techniques using an expression vector that encodes adefined fragment of SCCE, or by chemical synthesis. The ability of acandidate fragment to exhibit a characteristic of SCCE (e.g., binding toan antibody specific for SCCE) can be assessed by methods known in theart.

[0074] Purified SCCE or antigenic fragments of SCCE can be used togenerate new antibodies or to test existing antibodies (e.g., aspositive controls in a diagnostic assay) by employing standard protocolsknown to those skilled in the art. Included in this invention ispolyclonal antisera generated by using SCCE or a fragment of SCCE as theimmunogen in, e.g., rabbits. Standard protocols for monoclonal andpolyclonal antibody production known to those skilled in this art areemployed. The monoclonal antibodies generated by this procedure can bescreened for the ability to identify recombinant SCCE cDNA clones, andto distinguish them from other cDNA clones.

[0075] The invention encompasses not only an intact anti-SCCE monoclonalantibody, but also an immunologically-active antibody fragment, e.g., aFab or (Fab)₂ fragment; an engineered single chain Fv molecule; or achimeric molecule, e.g., an antibody which contains the bindingspecificity of one antibody, e.g., of murine origin, and the remainingportions of another antibody, e.g., of human origin.

[0076] In one embodiment, the antibody, or a fragment thereof, may belinked to a toxin or to a detectable label, e.g., a radioactive label,non-radioactive isotopic label, fluorescent label, chemiluminescentlabel, paramagnetic label, enzyme label, or colorimetric labelwell-known in the art. Examples of suitable toxins include diphtheriatoxin, Pseudomonas exotoxin A, ricin, and cholera toxin. Examples ofsuitable enzyme labels include alkaline phosphatase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, etc.Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I, ³²P,³⁵S, ¹⁴C, etc.

[0077] Paramagnetic isotopes for purposes of in vivo diagnosis can alsobe used according to the methods of this invention. There are numerousexamples of elements that are useful in magnetic resonance imaging. Fordiscussions on in vivo nuclear magnetic resonance imaging, see, forexample, Schaefer et al., (1989) JACC 14:472-480; Shreve et al., (1986)Magn. Reson. Med. 3:336-340; Wolf, G. L., (1984) Physiol. Chem. Phys.Med. NMR 16:93-95; Wesbey et al., (1984) Physiol. Chem. Phys. Med. NMR16:145-155; Runge et al., (1984) Invest. Radiol. 19:408-415. Examples ofsuitable fluorescent labels include a fluorescein label, anisothiocyalate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an ophthaldehyde label, afluorescamine label, etc. Examples of chemiluminescent labels include aluminal label, an isoluminal label, an aromatic acridinium ester label,an imidazole label, an acridinium salt label, an oxalate ester label, aluciferin label, a luciferase label, an aequorin label, etc.

[0078] Those of ordinary skill in the art will know of other suitablelabels which may be employed in accordance with the present invention.The binding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known and used by thoseof ordinary skill in the art. Typical techniques are described byKennedy et al., (1976) Clin. Chim. Acta 70, 1-31; and Schurs et al.,(1977) Clin. Chim.

[0079] Acta 81, 1-40. Coupling techniques mentioned in the latter arethe glutaraldehyde method, the periodate method, the dimaleimide method,the m-maleimidobenzyl-N-hydroxy-succinimide ester method. All of thesemethods are incorporated by reference herein.

[0080] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion. One skilled in the art will appreciatereadily that the present invention is well adapted to carry out theobjects and obtain the ends and advantages mentioned, as well as thoseobjects, ends and advantages inherent herein. Changes therein and otheruses which are encompassed within the spirit of the invention as definedby the scope of the claims will occur to those skilled in the art.

EXAMPLE 1

[0081] Amplification of Serine Proteases Using Redundant and SpecificPrimers

[0082] Only cDNA preparations deemed free of genomic DNA were used forgene expression analysis. Redundant primers were prepared for serineproteases, metallo-proteases and cysteine protease. The primers weresynthesized to consensus sequences of amino acid surrounding thecatalytic triad for serine proteases, viz. histidine . . . aspartate . .. and serine. The sequences of both sense (histidine & aspartate) andantisense (aspartate and serine) redundant primers are shown in Table 2.TABLE 2 PCR Primers 5′→3′ SEQ ID No. Redundant Primers: Serine Protease(histidine) = S1 tgggtigtiacigcigcica(ct)tg 1 Serine Protease (asparticacid) = AS1 a(ag)ia(ag)igciatitcitticc 2 Serine Protease (serine) = AS11a(ag)iggiccicci(cg)(ta)(ag)tcicc 3 Cysteine Protease - senseca(ag)ggica(ag)tg(ct)ggi(ta)(cg)itg(ct)tgg 4 Cysteine Protease -antisense taiccicc(ag)tt(ag)caicc(ct)tc 5 Metallo Protease - sensecci(ac)gitg(tc)ggi(ga)(ta)icciga 6 Metallo Protease - antisensett(ag)tgicciai(ct)tc(ag)tg 7 Specific Primers: Serine Protease (SCCE) =sense tgtcccgatggcgagtgttt 8 Serine Protease (SCCE) = antisensecctgttggccatagtactgc 9 Serine Protease (SCCE) = senseagatgaatgagtacaccgtg 10 Serine Protease (SCCE) = antisenseccagtaagtccttgtaaacc 11 Serine Protease (Comp B) = senseaagggacacgagagctgtat 12 Serine Protease (Comp B) = antisenseaagtggtagttggaggaagc 13 Serine Protease (Protease M) = sensectgtgatccaccctgactat 20 Serine Protease (Protease M) = antisensecaggtggatgtatgcacact 21 Serine Protease (TADG12) = sense (Ser 10-s)gcgcactgtgtttatgagat 22 Serine Protease (TADG12) = antisense (Ser10-as)ctctttggcttgtacttgct 23 Serine Protease (TADG13) = sensetgagggacatcattatgcac 24 Serine Protease (TADG13) = antisensecaagttttccccataattgg 25 Serine Protease (TADG14) = senseacagtacgcctgggagacca 26 Serine Protease (TADG14) = antisensectgagacggtgcaattctgg 27 Cysteine Protease (Cath-L) = senseattggagagagaaaggctac 14 Cysteine Protease (Cath-L) = antisensecttgggattgtacttacagg 15 Metallo Protease (PUMP1) = sensecttccaaagtggtcacctac 16 Metallo Protease (PUMP1) = antisensectagactgctaccatccgtc 17

EXAMPLE 2

[0083] Carcinoma Tissue

[0084] Several protease entities were identified and subcloned from PCRamplification of CDNA derived from serous cystadenocarcinomas.Therefore, the proteases described herein are reflective of surfaceactivities for this type of carcinoma, the most common form of ovariancancer. It was also shown that PCR amplification bands unique to themucinous tumor type and the clear cell type have similar base pair size.About 20-25% of ovarian cancers are classified as either mucinous, clearcell, or endometrioid.

EXAMPLE 3

[0085] Ligation, Transformation and Sequencing

[0086] To determine the identity of the PCR products, all theappropriate bands were ligated into Promega T-vector plasmid and theligation product was used to transform JM109 cells (Promega) grown onselective media. After selection and culturing of individual colonies,plasmid DNA was isolated by means of the WIZARD MINIPREP™ DNApurification system (Promega). Inserts were sequenced using a PrismReady Reaction Dydeoxy Terminators cycle sequencing kit (AppliedBiosystems). Residual dye terminators were removed from the completedsequencing reaction using a CENTRISEP SPIN™ column (PrincetonSeparation), and samples were loaded into an Applied Biosystems Model373A DNA sequencing system. The results of subcloning and sequencing forthe serine protease primers are summarized in Table 3. TABLE 3 SerineProtease Candidates Subclone Primer Set Gene Candidate 1 His-Ser SCCE 2His-Ser SCCE 3 His-Ser Compliment B 4 His-Asp Cofactor 1 5 His-AspTADG-12* 6 His-Ser TADG-13* 7 His-Ser TADG-14* 8 His-Ser Protease M 9His-Ser TADG-15*

EXAMPLE 4

[0087] Cloning and Characterization

[0088] Cloning and characterization of new gene candidates wasundertaken to expand the panel representative of extracellular proteasesspecific for ovarian carcinoma subtypes. Sequencing of the PCR productsderived from tumor cDNA confirms the potential candidacy of these genes.The three novel genes all have conserved residues within the catalytictriad sequence consistent with their membership in the serine proteasefamily.

[0089] PCR products amplified from normal and carcinoma cDNAs werecompared using sense-histidine and antisense-aspartate as well assense-histidine and antisense-serine. The anticipated PCR products ofapproximately 200 bp and 500 bp for those pairs of primers were observed(aspartate is approximately 50-70 amino acids downstream from histidine,and serine is about 100-150 amino acids toward the carboxy end fromhistidine).

[0090]FIG. 1 shows a comparison of PCR products derived from normal andcarcinoma cDNA as shown by staining in an agarose gel. Two distinctbands in Lane 2 were present in the primer pair sense-His/antisense ASP(AS1) and multiple bands of about 500 bp are noted in the carcinoma lanefor the sense-His/antisense-Ser (AS2) primer pairs in Lane 4.

EXAMPLE 5

[0091] Ouantitative PCR

[0092] The mRNA overexpression of SCCE was detected and determined usingquantitative PCR. Quantitative PCR was performed generally according tothe method of Noonan et al. (1990). The following oligonucleotideprimers were used: SCCE: forward 5′-AGATGAATGAGTACACCGTG-3′, (SEQ ID No.10) and reverse 5′-CCAGTAAGTCCTTGTAAACC-3′; (SEQ ID No. 11) andβ-tubulin: forward 5′- TGCATTGACAACGAGGC -3′, (SEQ ID No. 18) andreverse 5′-CTGTCTTGACATTGTTG-3′. (SEQ ID No. 19)

[0093] β-tubulin was utilized as an internal control. The predictedsizes of the amplified genes were 339 bp for SCCE and 454 bp forβ-tubulin. The primer sequences used in this study were designedaccording to the cDNA sequences described by Hansson et al. (1994) forSCCE, and Hall et al. (1983) for β-tubulin. The PCR reaction mixtureconsisted of cDNA derived from 50 ng of mRNA converted by conventionaltechniques, 5 pmol of sense and antisense primers for both the SCCE geneand the β-tubulin gene, 200 μmol of dNTPs, 5 μCi of [α-³²P]dCTP and 0.25units of Taq DNA polymerase with reaction buffer (Promega) in a finalvolume of 25 μl. The target sequences were amplified in parallel withthe β-tubulin gene. Thirty cycles of PCR were carried out in a ThermalCycler (Perkin-Elmer Cetus). Each cycle of PCR included 30 sec ofdenaturation at 95° C, 30 sec of annealing at 63° C. and 30 sec ofextension at 72° C. It was previously established and confirmed for SCCEthat co-amplification with β-tubulin under these conditions for 30cycles remain linear for both products.

[0094] The PCR products were separated on 2% agarose gels and theradioactivity of each PCR product was determined by using a PhosphoImager (Molecular Dynamics). In the present study, expression of SCCEwas calculated as the ratio (SCCE/β-tubulin) as measured byphosphoimager. The overexpression cut-off value was defined as the meanvalue for normal ovary +2SD. The student's t test was used for thecomparison of the mean values of normal ovary and tumors.

[0095] Experiments comparing PCR -amplification in normal ovary andovarian carcinoma suggested overexpression and/or alteration in mRNAtranscript in tumor tissues. Northern blot analysis of TADG-14 confirmsa transcript size of 1.4 kb and data indicate overexpression in ovariancarcinoma (FIG. 2). Isolation and purification using both PCR and aspecific 250 bp PCR product to screen positive plaques yielded a 1.2 kbclone of TADG-14. Other proteases were amplified by the same methodusing the appropriate primers from Table 2.

EXAMPLE 6

[0096] Tissue Bank

[0097] A tumor tissue bank of fresh frozen tissue of ovarian carcinomasas shown in Table 4 was used for evaluation. Approximately 100 normalovaries removed for medical reasons other than malignancy were obtainedfrom surgery and were available as controls. TABLE 4 Ovarian CancerTissue Bank Total Stage I/11 Stage III/IV No Stage Serous Malignant 16615 140 8 LMP 16 9 7 0 Benign 12 0 0 12 Mucinous Malignant 26 6 14 6 LMP28 25 3 0 Benign 3 0 0 3 Endometrioid Malignant 38 17 21 0 LMP 2 2 0 0Benign 0 0 0 0 Other* Malignant 61 23 29 9 LMP 0 0 0 0 Benign 5 0 0 5

[0098] From the tumor bank, approximately 100 carcinomas were evaluatedencompassing most histological sub-types of ovarian carcinoma, includingborderline or low-malignant potential tumors and overt carcinomas. Theapproach included using mRNA prepared from fresh frozen tissue (bothnormal and malignant) to compare expression of genes in normal, lowmalignant potential tumors and overt carcinomas. The cDNA prepared frompolyA+mRNA was deemed to be genomic DNA free by checking allpreparations with primers that encompassed a known intron-exon splicesite using both β-tubulin and p53 primers.

EXAMPLE 7

[0099] Northern Blots Analvsis

[0100] Significant information can be obtained by examining theexpression of these candidate genes by Northern blot. Analysis of normaladult multi-tissue blots offers the opportunity to identify normaltissues which may express the protease. Ultimately, if strategies forinhibition of proteases for therapeutic intervention are to bedeveloped, it is essential to appreciate the expression of these genesin normal tissue if and when it occurs.

[0101] Northern panels for examining expression of genes in amulti-tissue normal adult as well as fetal tissue are commerciallyavailable (CLONTECH). Such evaluation tools are not only important toconfirm the overexpression of individual transcripts in tumor versusnormal tissues, but also provides the opportunity to confirm transcriptsize, and to determine if alternate splicing or other transcriptalteration may occur in ovarian carcinoma.

[0102] Northern blot analysis was performed as follows: 10 μg of mRNAwas loaded onto a 1% formaldehyde-agarose gel, electrophoresed andblotted onto a HyBond-N⁺™ nylon membrane (Amersham). ³²P-labeled cDNAprobes were made using Prime-a-Gene Labeling System™ (Promega). The PCRproducts amplified by specific primers were used as probes. Blots wereprehybridized for 30 min and then hybridized for 60 min at 68° C. with³²P-labeled cDNA probe in ExpressHyb™ Hybridization Solution (CLONTECH).Control hybridization to determine relative gel loading was accomplishedusing the β-tubulin probe.

[0103] Normal human tissues including spleen, thymus, prostate, testis,ovary, small intestine, colon, peripheral blood leukocyte, heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas and normalhuman fetal tissues (Human Multiple Tissue Northern Blot; CLONTECH) wereall examined using the same hybridization procedure.

EXAMPLE 8

[0104] PCR Products Corresponding to Serine Cysteine and Metallo-Proteases

[0105] Based on their unique expression in either low malignantpotential tumors or carcinomas, PCR-amplified cDNA products were clonedand sequenced and the appropriate gene identified based upon nucleotideand amino acid sequences stored in the GCG and EST databases. FIGS. 3, 4& 5 show the PCR product displays comparing normal and carcinomatoustissues using redundant primers for serine proteases (FIG. 3), forcysteine proteases (FIG. 4) and for metallo-proteases (FIG. 5). Note thedifferential expression in the carcinoma tissues versus the normaltissues. The proteases were identified using redundant cDNA primers (seeTable 2) directed towards conserved sequences that are associated withintrinsic enzyme activity (for serine proteases, cysteine proteases andmetallo-proteases) by comparing mRNA expression in normal, low malignantpotential and overt ovarian carcinoma tissues according to Sakanari etal. (1989).

EXAMPLE 9

[0106] Serine Proteases

[0107] For the serine protease group, using the histidine domain primersense, S1, in combination with antisense primer AS2, the followingproteases were identified:

[0108] (a) Hepsin, a trypsin-like serine protease cloned from hepatomacells shown to be a cell surface protease essential for the growth ofhepatoma cells in culture and highly expressed in hepatoma tumor cells(FIG. 3, Lane 4);

[0109] (b) Complement factor B protease (human factor IX), a proteaseinvolved in the coagulation cascade and associated with the productionand accumulation of fibrin split products associated with tumor cells(FIG. 3, Lane 4). Compliment factor B belongs in the family ofcoagulation factors X (Christmas factor). As part of the intrinsicpathway, compliment factor B catalyzes the proteolytic activation ofcoagulation factor X in the presence of Ca²⁺ phospholipid and factorVIIIa e5; and

[0110] (c) A stratum corneum chymotryptic enzyme (SCCE) serine proteaseinvolved in desquamation of skin cells from the human stratum corneum(FIG. 3, Lane 4). SCCE is expressed in keratinocytes of the epidermisand functions to degrade the cohesive structures in the cornified layerto allow continuous skin surface shedding.

EXAMPLE 10

[0111] Cysteine Proteases

[0112] In the cysteine protease group, using redundant sense andanti-sense primers for cysteine proteases, one unique PCR product wasidentified by overexpression in ovarian carcinoma when compared tonormal ovarian tissue (FIG. 4, Lanes 3-5).

[0113] Cloning and sequencing this PCR product identified a sequence ofCathepsin L, which is a lysomal cysteine protease whose expression andsecretion is induced by malignant transformation, growth factors andtumor promoters. Many human tumors (including ovarian) express highlevels of Cathepsin L. Cathepsin L cysteine protease belongs in thestromolysin family and has potent elastase and collagenase activities.Published data indicates increased levels in the serum of patients withmucinous cystadenocarcinoma of the ovary. It has not heretofore beenshown to be expressed in other ovarian tumors.

EXAMPLE 11

[0114] Metallo-proteases

[0115] Using redundant sense and anti-sense primers for themetallo-protease group, one unique PCR product was detected in the tumortissue which was absent in normal ovarian tissue (FIG. 5, Lanes 2-5).Subcloning and sequencing this product indicates it has completehomology in the appropriate region with the so-called PUMP-1 (MMP-7)gene. This zinc-binding metallo-protease is expressed as a proenzymewith a signal sequence and is active in gelatin and collagenasedigestion. PUMP-1 has also been shown to be induced and overexpressed in9 of 10 colorectal carcinomas compared to normal colon tissue,suggesting a role for this substrate in the progression of this disease.

EXAMPLE 12

[0116] mRNA Expression of SCCE in Ovarian Tumors

[0117] To evaluate mRNA expression of SCCE in ovarian tumors,semi-quantitative PCR was performed. A preliminary study confirmed thelinearity of the PCR amplification according to the methods of Shigemasaet al. (1997) and Hall et al. (1983).

[0118]FIG. 6 shows an example of comparative PCR using SCCE primersco-amplified with the internal control β-tubulin primers. Analysis ofthe data as measured using the phosphoimager and compared as ratios ofexpression (SCCE/β-tubulin) indicate that SCCE expression is elevated intumor cases 19, 14, 15, 16, 43, 23, 36 and 37 compared to that of normalovaries.

[0119] To confirm the results of the initial quantitative PCR and toexamine the size of the transcript, Northern blot hybridization wasperformed in representative cases of each histological type of carcinoma(FIG. 7A). Northern blot hybridization with a ³²P-labeled SCCE probe(nucleotides 232-570) revealed 1.2 kb and 2.0 kb transcripts, asreported previously in normal skin tissue (Hansson et al., 1994). Thosetumor cases which showed overexpression of SCCE by quantitative PCR alsoshowed intense bands of SCCE transcript expression by Northern blotanalysis including serous, mucinous, endometrioid and clear cellcarcinoma. No transcripts were detected in normal ovarian tissue (Lane1). Normal human tissues (spleen, thymus, prostate, testis, ovary, smallintestine, colon, peripheral blood leukocyte, heart, brain, placenta,lung, liver, skeletal muscle, kidney and pancreas) and normal humanfetal tissues (brain, lung, liver and kidney) examined by Northern blotanalysis showed no visible SCCE transcripts (FIGS. 7B & 7C). Blots fornormal human adult tissues and fetal tissues were subsequently probed toconfirm the presence of β-tubulin transcripts.

[0120] Table 5 summarizes the results of the evaluation of SCCEexpression in 10 individual normal ovarian tissues and 44 ovariancarcinomas. Overall, SCCE mRNA overexpression (overexpression=mean valuefor normal ovary+2SD) was found in 8 of 12 LMP tumors (66.7%) and 25 of32 carcinoma cases (78.1%) with p values of <0.05 and <0.001respectively (FIG. 8). Overexpression of SCCE transcripts was detectedin all ovarian carcinoma subtypes and in both early stage and late stagetumor samples. In the five cases where positive confirmation of lymphnode metastasis was identified, all five cases showed overexpression ofSCCE at a level of more than four standard deviations above the levelfor normal ovary. It should be noted that three of these tumors wereclassified as low malignant potential tumors (all serous adenomas)suggesting a possible relationship between the progression of earlystage disease to the lymph when overexpression of SCCE is manifest.TABLE 5 Patient Characteristics and Expression of SCCE Gene mRNAexpression^(c) Case Histological Type^(a) Stage/Grade  LN^(b) SCCE 1normal ovary n 2 normal ovary n 3 normal ovary n 4 normal ovary n 5normal ovary n 6 normal ovary n 7 normal ovary n 8 normal ovary n 9normal ovary n 10 normal ovary n 11 s adenoma (LMP) 1/1 n 4+ 12 sadenoma (LMP) 1/1 NE n 13 s adenoma (LMP) 1/1 NE 2+ 14 s adenoma (LMP)1/1 n 4+ 15 s adenoma (LMP) 3/1 p 4+ 16 s adenoma (LMP) 3/1 p 4+ 17 sadenoma (LMP) 3/1 p 4+ 18 m adenoma (LMP) 1/1 NE 4+ 19 m adenoma (LMP)1/1 n 4+ 20 m adenoma (LMP) 1/1 n n 21 m adenoma (LMP) 1/1 NE n 22 madenoma (LMP) 1/1 NE n 23 s carcinoma 1/2 n 4+ 24 s carcinoma 1/3 n 4+25 s carcinoma 3/1 NE 4+ 26 s carcinoma 3/2 NE 4+ 27 s carcinoma 3/2 p4+ 28 s carcinoma 3/2 NE 4+ 29 s carcinoma 3/3 NE 4+ 30 s carcinoma 3/3NE 4+ 31 s carcinoma 3/3 NE 4+ 32 s carcinoma 3/3 NE 4+ 33 s carcinoma3/3 n 4+ 34 s carcinoma 3/3 NE n 35 s carcinoma 3/3 NE 4+ 36 s carcinoma3/3 NE 4+ 37 s carcinoma 3/3 NE 4+ 38 s carcinoma 3/3 n 4+ 39 scarcinoma 3/2 NE 4+ 40 s carcinoma 3/3 NE 4+ 41 s carcinoma 3/2 NE n 42m carcinoma 1/2 n n 43 m carcinoma 2/2 NE 4+ 44 m carcinoma 2/2 n n 45 mcarcinoma 3/1 NE n 46 m carcinoma 3/2 NE n 47 m carcinoma 3/2 NE n 48 mcarcinoma 3/3 NE 4+ 49 e carcinoma 2/3 n 4+ 50 e carcinoma 3/2 NE 4+ 51e carcinoma 3/3 NE 4+ 52 c carcinoma 1/3 n 4+ 53 c carcinoma 1/1 n 4+ 54c carcinoma 3/2 p 4+

[0121] The expression ratio (mean value±SD) for normal ovary wasdetermined as 0.046±0.023, for LMP tumors as 0.405±0.468 and forcarcinoma as 0.532±0.824 (Table 6). From a histological point of view,overexpression of SCCE was observed in 23 of 26 serous tumors (88.5%)including 6 of 7 LMP tumors and 17 of 19 carcinomas. However only 4 of12 mucinous tumors (33.3%) including 2 of 5 LMP tumors and 2 of 7carcinomas showed overexpression of SCCE. For endometrioid and clearcell carcinoma, stratum corneum chymotrytic enzyme was found to beoverexpressed in all 6 cases (Table 6). TABLE 6 Overexpression of SCCEin Ovarian Carcinoma N Overexpression of SCCE Expression Ratio^(a)Normal 10  0 (0%) 0.046 ± 0.023 LMP 12  8 (66.7%) 0.405 ± 0.468 serous 7 6 (85.7%) 0.615 ± 0.518 mucinous 5  2 (40.0%) 0.111 ± 0.117 Carcinoma32 25 (78.1%) 0.532 ± 0.824 serous 19 17 (89.5%) 0.686 ± 1.027 mucinous7  2 (28.6%) 0.132 ± 0.265 endometrioid 3  3 (100%) 0.511 ± 0.205 clearcell 3  3 (100%) 0.515 ± 0.007

EXAMPLE 13

[0122] Western Blot Analysis

[0123] Polyclonal rabbit antibodies were generated by immunization witha combination of 2 poly-lysine linked multiple Ag peptides derived fromSCCE protein sequences PLQILLLSLALE (SEQ ID No. 28) and SFRHPGYSTQTH(SEQ ID No. 29). Approximately 20 ng of MDA-MBA-435S and HeLa celllysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDFat 100 V for 40 minutes at 4° C. The proteins were fixed to the membraneby incubation in 50% MeOH for 10 minutes. The membrane was blockedovernight in TBS, pH 7.8 containing 0.2% non-fat milk. Primary antibodywas added to the membrane at a dilution of 1:100 in 0.2% milk/TBS andincubated for 2 hours at room temperature. The blot was washed andincubated with a 1:3000 dilution of alkaline-phosphatase conjugated goatanti-rabbit IgG (BioRad) for one hour at room temperature. The blot waswashed and incubated with a chemiluminescent substrate before a 10second exposure to X-ray film for visualization.

[0124] Two cell lines HeLa and MDA-MB-435S previously shown to expressmRNA transcripts were examined by Western blot to confirm the presenceof SCCE protein. FIG. 9 indicates that polyclonal antibodies developedto peptides (12 mers bound to polylysine) derived from the amino andcarboxy termini of SCCE bind a protein of approximately 30 kDa incytosolic extracts of HeLa and MDA-MB-435S cells. The ovarian tumor cellline CAOV3 was also examined for SCCE expression and a protein productcould not be detected (data not shown). This molecular size proteinagrees with the anticipated and known parameters for the SCCE protein.It should be noted that only a single band was detected by Western blotanalysis of cystosolic protein. It might be anticipated that the SCCEprotein prior to secretion would be present in the inactivated parentform i.e. the seven amino terminal peptide removed on activation wouldstill be present on the enzyme. In this pre-active form of the enzyme itwould be anticipated that the apparent molecular weight on Western blotwould be about 30 kDa.

EXAMPLE 14

[0125] Immunohistochemistry

[0126] Immunohistochemical localization of SCCE antigen was examinedusing normal ovaries, mucinous LMP tumor and adenocarcinomas (includingserous adenocarcinomas, mucinous adenocarcinoma and clear cellcarcinomas) in the same series of the samples for mRNA isolation.Formalin fixed and paraffin embedded sections, 4 μm thick, were cut andmounted on aminopropyltriethoxysilane treated slides. Slides wereroutinely deparaffinized with xylene and rehydrated with a series ofethanol washes. Nonenzymatic antigen retrieval was performed byprocessing using microwave heat treatment in 0.01 M sodium citratebuffer (pH 6.0). Immunohistochemical staining was performed manuallyusing the avidin-biotin peroxidase complex technique (Vectastain EliteABC kit, Vector Laboratories). Anti-SCCE rabbit polyclonal antibody wasgenerated by immunization with a combination of 2 poly-lysine linkedmultiple Ag peptide derived from the SCCE protein-sequences.

[0127] This indirect immunoperoxidase staining procedure was performedat room temperature. Endogenous peroxidase and nonspecific backgroundstaining were blocked by incubating slides with methanol with 0.3% H₂0₂for 30 minutes. After washing with phosphate-buffered saline (PBS) for10 minutes, sections were incubated with biotinylated anti-rabbit IgGfor 30 minutes. After washing with PBS for 10 minutes, slides wereincubated with ABC reagent for 30 minutes. The final products werevisualized by using AEC substrate system (DAKO Corporation) and sectionswere counterstained with Mayer hematoxylin for 20 seconds beforemounting. Positive controls and negative controls were used for eachsection. Negative controls were performed by using normal rabbit seruminstead of the primary antibody. All experiments were duplicated. Thestained slides were examined microscopically by 3 observers. More than10% of positive tumor cells was the criterion for a 1+ positive stainingand more than 50% of positive tumor cells was the criterion for a 2+positive staining.

[0128] To further confirm the presence of the SCCE protein in ovariantumor cells as opposed to its elaboration by supporting stromal or bloodvessel cells, both normal ovarian epithelia and ovarian tumor tissuewere examined by immunohistochemistry using the polyclonal antiserumdescribed above. All 14 ovarian tumors showed positive staining of SCCE,whereas normal ovarian surface epithelium showed very weak expression ofSCCE antigen (FIG. 10A). 8 of 10 serous adenocarcinomas, 1 of 1 mucinousadenocarcinoma, and 2 of 2 clear cell carcinomas showed 2+ positivestaining (more than 50% of positive tumor cells) of SCCE (Table 7).FIGS. 10C and 10E show that stratum corneum chymotrytic enzyme stainingis localized to the cytoplasm and the cell membrane of ovarian tumorcells. The negative control of each case was also performed, wherein theresult showed no nonspecific staining of stratum corneum chymotryticenzyme (FIG. 10B, 10D and 10F). Staining of normal ovarian epithelialcells showed little SCCE expression (FIG. 10A). TABLE 7Immunohistochemical Expression of SCCE Protein in Normal Ovary andOvarian Tumor Lab No. Histology SCCE normal ovary weak + normal ovaryweak + normal ovary weak + normal ovary weak + normal ovary weak +normal ovary weak + 1036 mucinous LMP + 475 serous carcinoma + 465serous carcinoma ++ 464 serous carcinoma ++ 1039 serous carcinoma ++ 960serous carcinoma ++ 962 serous carcinoma ++ 1551 serous carcinoma ++1813 serous carcinoma ++ 1817 serous carcinoma + 1819 serous carcinoma++ 1244 mucinous carcinoma ++ 947 clear cell carcinoma ++ 948 clear cellcarcinoma ++

EXAMPLE 15

[0129] Summary of Proteases Detected Herein

[0130] Most of the above-listed proteases were identified from thesense-His/antisense-Ser primer pair, yielding a 500 bp PCR product (FIG.1, Lane 4). Some of the enzymes are familiar, a short summary of eachfollows.

[0131] Hepsin

[0132] Hepsin is a trypsin-like serine protease cloned from hepatomacells. Hepsin is an extracellular protease (the enzyme includes asecretion signal sequence) which is anchored in the plasma membrane byits amino terminal domain, thereby exposing its catalytic domain to theextracellular matrix. Hepsin has also been shown to be expressed inbreast cancer cell lines and peripheral nerve cells. Hepsin has neverbefore been associated with ovarian carcinoma. Specific primers for thehepsin gene were synthesized and the expression of hepsin examined usingNorthern blots of fetal tissue and ovarian tissue (both normal andovarian carcinoma).

[0133]FIG. 11A shows that hepsin was expressed in ovarian carcinomas ofdifferent histologic types, but not in normal ovary. FIG. 11B shows thathepsin was expressed in fetal liver and fetal kidney as anticipated, butat very low levels or not at all in fetal brain and lung. FIG. 11C showsthat hepsin overexpression is not observed in normal adult tissue.Slight expression above the background level is observed in the adultprostate. The mRNA identified in both Northern blots was the appropriatesize for the hepsin transcript.

[0134] The expression of hepsin was examined in 10 normal ovaries and 44ovarian tumors using specific primers to β-tubulin and hepsin in aquantitative PCR assay.

[0135] Expression is presented as the ratio of ³²P-hepsin band to theinternal control, the ³²P-β-tubulin band. Hepsin mRNA is highlyoverexpressed in most histopathologic types of ovarian carcinomasincluding some low malignant potential tumors (see FIGS. 12A & 12B).Most noticeably, hepsin is highly expressed in serous, endometrioid andclear cell tumors tested. It is highly expressed in some mucinoustumors, but it is not overexpressed in the majority of such tumors.

[0136] Stratum corneum chymotrypsin enzyme (SCCE)

[0137] The PCR product identified was the catalytic domain of thesense-His/antisense-Ser of the SCCE enzyme. This extracellular proteasewas cloned, sequenced and shown to be expressed on the surface ofkeratinocytes in the epidermis. SCCE is a chymotrypsin-like serineprotease whose function is suggested to be in the catalytic degradationof intercellular cohesive structures in the stratum corneum layer of theskin. This degradation allows continuous shedding (desquamation) ofcells from the skin surface. The subcellular localization of SCCE is inthe upper granular layer in the stratum corneum of normalnon-palmoplantar skin and in the cohesive parts of hypertrophic plantarstratum corneum. SCCE is exclusively associated with the stratum corneumand has not been shown to be expressed in any carcinomatous tissues.

[0138] Northern blots were probed with the PCR product to determineexpression of SCCE in fetal tissue and ovarian carcinoma (FIGS. 7A, 7Band 7C). Noticeably, detection of SCCE messenger RNA on the fetalNorthern was almost non-existent (a problem with the probe or the blotwas excluded by performing the proper controls). A faint band appearedin fetal kidney. On the other hand, SCCE mRNA is abundant in the ovariancarcinoma mRNA (FIG. 7A). Two transcripts of the correct size areobserved for SCCE. The same panel of cDNA used for SCCE analysis wasused for SCCE expression.

[0139] No SCCE expression was detected in the normal ovary lane of theNorthern blot. A comparison of all candidate genes, including a loadingmarker (β-tubulin), was shown to confirm that this observation was not aresult of a loading bias. Quantitative PCR using SCCE primers, alongwith β-tubulin internal control primers, confirmed the overexpression ofSCCE mRNA in carcinoma of the ovary with no expression in normal ovariantissue (FIG. 6). FIG. 8 shows the ratio of SCCE to the β-tubulininternal standard in 10 normal and 44 ovarian carcinoma tissues. Again,it is observed that SCCE is highly overexpressed in ovarian carcinomacells. It is also noted that some mucinous tumors overexpress SCCE, butthe majority do not.

[0140] Protease M

[0141] Protease M was identified from subclones of the His—ser primerpair. This protease was cloned by Anisowicz, et al., and shown to beoverexpressed in carcinomas. A evaluation indicates that this enzyme isoverexpressed in ovarian carcinoma (FIG. 13).

[0142] Cofactor I and Complement factor B

[0143] Several serine proteases associated with the coagulation pathwaywere also subcloned. Examination of normal and ovarian carcinomas byquantitative PCR for expression of these enzymes, it was noticeable thatthis mRNA was not clearly overexpressed in ovarian carcinomas whencompared to normal ovarian tissue. It should be noted that the samepanel of tumors was used for the evaluation of each candidate protease.

EXAMPLE 16

[0144] Summary of Previously Unknown Proteases Detected Herein

[0145] TADG-12

[0146] TADG-12 was identified from the primer pairs,sense-His/antisense-Asp (see FIG. 1, Lanes 1 & 2). Upon subcloning bothPCR products in lane 2, the 200 bp product had a unique protease-likesequence not included in GenBank. This 200 bp product contains many ofthe conserved amino acids common for the His-Asp domain of the family ofserine proteins. The second and larger PCR product (300 bp) was shown tohave a high degree of homology with TADG-12 (His-Asp sequence), but alsocontained approximately 100 bp of unique sequence. Synthesis of specificprimers and the sequencing of the subsequent PCR products from threedifferent tumors demonstrated that the larger PCR product (present inabout 50% of ovarian carcinomas) includes an insert of about 100 bp nearthe 5′ end (and near the histidine) of the sequence. This insert may bea retained genomic intron because of the appropriate position of splicesites and the fact that the insert does not contain an open readingframe (see FIG. 14). This suggests the possibility of a splice sitemutation which gives rise to retention of the intron, or a translocationof a sequence into the TADG-12 gene in as many as half of all ovariancarcinomas.

[0147] TADG-13 and TADG-14

[0148] Specific primers were synthesized for TADG-13 and TADG-14 toevaluate expression of genes in normal and ovarian carcinoma tissue.Northern blot analysis of ovarian tissues indicates the transcript forthe TADG-14 gene is approximately 1.4 kb and is expressed in ovariancarcinoma tissues (FIG. 15A) with no noticeable transcript presence innormal tissue. In quantitative PCR studies using specific primers,increased expression of TADG- 14 in ovarian carcinoma tissues was notedcompared to a normal ovary (FIG. 15B). The presence of a specific PCRproduct for TADG-14 in both an HeLa library and an ovarian carcinomalibrary was also confirmed. Several candidate sequences corresponding toTADG-14 have been screened and isolated from the HeLa library.

[0149] Clearly from sequence homology, these genes fit into the familyof serine proteases. TADG-13 and TADG-14 are, however, heretoforeundocumented genes which the specific primers of the invention allow tobe evaluated in normal and tumor cells, and with which the presence orabsence of expression of these genes is useful in the diagnosis ortreatment selection for specific tumor types.

[0150] PUMP-1

[0151] In a similar strategy using redundant primers to metal bindingdomains and conserved histidine domains, a differentially expressed PCRproduct identical to matrix metallo-protease 7 (MMP-7) was identified,herein called PUMP-1. Using specific primers for PUMP-1, PCR produced a250 bp product for Northern blot analysis.

[0152] MMP-7 or PUMP-1 is differentially expressed in fetal lung andkidney tissues. FIG. 16A compares PUMP-1 expression in normal ovary andcarcinoma subtypes using Northern blot analysis. Notably, PUMP-1 isexpressed in ovarian carcinoma tissues, and again, the presence of atranscript in normal tissue was not detected. FIG. 16B shows theexpression of PUMP-1 in human fetal tissue, while no transcript could bedetected in either fetal brain or fetal liver. FIG. 16C shows thatPUMP-1 overexpression is not observed in normal adult tissue.Quantitative PCR comparing normal versus ovarian carcinoma expression ofthe PUMP-1 mRNA indicates that this gene is highly expressed in serouscarcinomas, including most low malignant serous tumors, and is, again,expressed to a lesser extent in mucinous tumors (FIGS. 17A & 17B).PUMP-1, however, is so far the protease most frequently foundoverexpressed in mucinous tumors (See Table 8 below).

[0153] Cathepsin-L

[0154] Using redundant cysteine protease primers to conserved domainssurrounding individual cysteine and histidine residues, the cathepsin-Lprotease was identified in several serous carcinomas. An initialexamination of the expression of cathepsin L in normal and ovarian tumortissue indicates that transcripts for the cathepsin-L protease arepresent in both normal and tumor tissues (FIG. 18). However, itspresence or absence in combination with other proteases of the presentinvention permits identification of specific tumor types and treatmentchoices.

[0155] Conclusion

[0156] Redundant primers to conserved domains of serine, metallo-, andcysteine proteases have yielded a set of genes whose mRNAs areoverexpressed in ovarian carcinoma. The genes which are clearlyoverexpressed include the serine proteases hepsin, SCCE, protease M,TADG12, TADG14 and the metallo-protease PUMP-1 (see FIG. 19 and Table8). Northern blot analysis of normal and ovarian carcinoma tissuesindicated overexpression of hepsin, SCCE, PUMP-1 and TADG-14. Aβ-tubulin probe to control for loading levels was included. TABLE 8Overexpression of Proteases in Ovarian Tumors Type N Hepsin SCCE Pump-1Protease M Normal 10   0% (0/10)   0% (0/10)   0% (0/10)   0% (0/10) LMP12 58.3% (7/12) 66.7% (8/12) 75.0% (9/12)   75% (9/12) serous 7 85.7%(6/7) 85.7% (6/7) 85.7% (6/7)  100% (7/7) mucinous 5 20.0% (1/5) 40.0%(2/5)   60% (3/5) 40.0% (2/5) Carcinoma 32 84.4% (27/32) 78.1% (25/32)81.3% (26/32) 90.6% (29/32) serous 19 94.7% (18/19) 89.5% (17/19) 78.9%(15/19) 94.7% (18/19) mucinous 7 42.9% (3/7) 28.6% (2/7) 71.4% (5/7)85.7% (6/7) endometr. 3  100% (3/3)  100% (3/3)  100% (3/3)  100% (3/3)clear cell 3  100% (3/3)  100% (3/3)  100% (3/3) 67.7% (2/3)

[0157] Discussion

[0158] For the most part, these proteins previously have not beenassociated with the extracellular matrix of ovarian carcinoma cells. Nopanel of proteases which might contribute to the growth, shedding,invasion and colony development of metastatic carcinoma has beenpreviously described, including the three new candidate serine proteaseswhich are herein disclosed. The establishment of an extracellularprotease panel associated with either malignant growth or malignantpotential offers the opportunity for the identification of diagnostic orprognostic markers and for therapeutic intervention through inhibitionor down regulation of these proteases.

[0159] The availability of the instant gene-specific primers coding forthe appropriate region of tumor specific proteases allows for theamplification of a specific cDNA probe using Northern and Southernanalysis, and their use as markers to detect the presence of the cancerin tissue. The probes also allow more extensive evaluation of theexpression of the gene in normal ovary versus low malignant potentialtumor, as well as both high- and low-stage carcinomas. The evaluation ofa panel of fresh frozen tissue from all the carcinoma subtypes (Table 4)allowed the determination of whether a protease is expressedpredominantly in early stage disease or within specific carcinomasubtypes. It was also determined whether each gene's expression isconfined to a particular stage in tumor progression and/or is associatedwith metastatic lesions. Detection of specific combinations of proteasesis an identifying characteristic of the specific tumor types and yieldsvaluable information for diagnoses and treatment selection. Particulartumor types may be more accurately diagnosed by the characteristicexpression pattern of each specific tumor.

[0160] Specifically, the present invention utilizes primers to theconserved catalytic triad domain of the serine protease family (viz.His—Asp—Ser). Using such a strategy to display serine proteasetranscripts found in abundance in carcinoma tissues, with little or noexpression in normal ovary, SCCE gene was detected.

[0161] The overall expectation of the search was to identify cellsurface or secreted products which may promote either tumor growth ormetastasis. Confirmation of the presence of SCCE (a secreted serineprotease) in ovarian tumors was indicated initially by subcloning andsequencing PCR products derived from amplification of tumor cDNA usingredundant primes to the histidine (sense) and the serine (antisense)conserved domains of the serine protease catalytic sequences.Characterization of the SCCE protease (Egelrud, 1993) indicated that thecohesion between individual comeocytes in the stratum comeurn, theprimary substrate for cellular desquamation or shedding of skin cellsmay be degraded by SCCE. Proteolysis of these intercellular matrices isone of the major events preceding desquamation. SCCE has only beenidentified in the stratum comeurn (Egelrud, 1993; Hansson et al., 1994)and immunohistochemical studies confirmed its unique tissue specificexpression by the epithelial cells of the stratum comeurn (SondeIl etal., 1994). It was therefore surprising to discover that this highlyconserved expression of SCCE to skin is obviated when transformation andcarcinogenesis of ovarian epithelial cells occurs. The clearlydistinctive pattern of expression in both low malignant potential tumorsand overt carcinomas of the ovary over normal ovarian tissue suggeststhat the SCCE protease may also play a role in shedding or desquamationof ovarian tumor cells. This association is especially well preserved inserous adenocarcinomas where disease progression is characterized byearly foci of peritoneal metastasis and which may be the result of anearly overexpression of enzymes such as SCCE and consequent tumor cellshedding. Because SCCE and other proteases (e.g. hepsin) areoverexpressed in ovarian tumors (again with particularly highoverexpression in serous tumors), it seems likely that a concert oflytic activity at the cell surface may be involved in malignantpotential. Several aspects of the tumorigenic process can be dissectedand identified as component parts of such a surface protease concertviz 1) initial expansion of newly transformed cells into the surroundingmatrix of supporting tissue of the primary organ; 2) desquamation orshedding of tumor cells into the surrounding environment; 3) invasion ofbasement membrane of the target organ of metastasis; and 4) activationof mitogenic and angiogenic factors to support the newly establishedmetastatic colony.

[0162] While it is not yet clear which proteases are the primary agentsin each of these malignant progression steps, the data here indicate thepotential for the involvement of SCCE in the shedding or desquamationphase of this progression. Certain other factors remain to be resolvedeven with regard to SCCE involvement in tumor cell shedding whichinclude activation of SCCE by proteolysis or cleaving of theaminoterminal peptide of the pro-protease. Furthermore, anantileukoprotease which specifically inhibits SCCE activity has beenrecently identified (Wiedow, O. (1995) Isolierung und Charakterisierungvon Serinprotease Inhibitoren der menschlichen Epidermis, Köster,Berlin). The presence of such an inhibitor might effectively inhibitshedding or desquamation of tumor cells as it has been shown to inhibitthe detachment of comeocytes of keratinized skin tissue.

[0163] While there remains an intricate interaction between surfaceprotease expression/activation and/or inhibition, it appears likely thata concert of enzymes which contribute to tumor growth and spread providea mechanism for such a progression. SCCE expression on ovarian tumorcell surfaces can provide one mechanism by which tumor cells may be shedearly in the tumor progression process of serous carcinomas.

[0164] The unique presence of this protease to keratinized stratumcorneum and the present data showing lack of transcript presence in allnormal adult and fetal tissues examined support the potential of thissecreted extracellular enzyme as a useful marker for ovarian carcinoma.The fact that inhibition of such an activity prevents normaldesquamation of skin cells also points to the potential of SCCE as atarget for inhibition or down regulation in therapeutic intervention inthe spread or metastasis of ovarian carcinoma.

EXAMPLE 17

[0165] SCCE Peptides As Target Epitopes For Human CD8⁺ Cytotoxic T Cells

[0166] Two computer programs were used to identify 9-mer peptidescontaining binding motifs for HLA class I molecules. The first, based ona scheme devised by Parker et al (1994), was developed by theBioinformatics and Molecular Analysis Section (BIMAS) of the Center forInformation Technology, NIH, and the second, known as SYFPEITHI, wasformulated by Rammensee and colleagues at the University of Tubingen,Germany.

[0167] Peptides that possessed HLA A2.1 binding motifs were synthesizedand tested directly for their ability to bind HLA A2.1. This techniqueemploys T2 cells which are peptide transporter-deficient and thusexpress low endogenous HLA class I levels due to inability to loadpeptide and stabilize HLA class I folding for surface expression. It hasbeen showed that addition of exogenous peptides capable of binding HLAA2. 1 (A*0201) could increase the number of properly folded HLA A2.1molecules on the cell surface, as revealed by flow cytometry (Nijman etal, 1993).

[0168] Monocyte-derived DC were generated from peripheral blood drawnfrom normal adult donors of the appropriate HLA type. Adherent monocyteswere cultured in AIM-V (Gibco-BRL) supplemented with GM-CSF and IL-4according to standard techniques (Santin et al, 2000). After 5-6 days,DC maturation was induced by addition of PGE₂, IL-1b and TNFa for afurther 48 h.

[0169] Mature DC were loaded with peptide (2×10⁶ DC with 50 mg/mlpeptide in 1 ml serum-free AIM-V medium for 2 h at 37° C.) and washedonce prior to culture with 1×10⁶/ml peripheral blood mononuclear cells(PBMC) in AIM-V or AIM-V plus 5% human AB serum. The PBMC:DC ratio wasbetween 20:1 and 30:1. After 7 days, responder T cells were restimulatedwith peptide-loaded, irradiated autologous DC or PBMC atresponder:stimulator ratios between 10:1 and 20:1 or 1:1 and 1:10respectively.

[0170] At this point, cultures were supplemented with recombinant humanIL-2 (10-100 U/ml), and fed with 50-75% changes of fresh medium plusIL-2 every 2-4 days. T cell lines were established and maintained bypeptide restimulation every 14-21 days. Responder CD8⁺ T cells werepurified by positive selection with anti-CD8-coupled magnetic beads(Dynal, Inc.) after the 2^(nd) or 3^(rd) antigen stimulation.

[0171] Peptide-specific cytotoxicity was tested in standard 5-6 hmicrowell ⁵¹Cr-release assays (Nazaruk et al, 1998). AutologousEBV-transformed lymphoblastoid cell lines (LCL) were loaded with peptide(50 mg/ml, 1 h at 37° C.) and subsequently ⁵¹Cr-labeled (50 mCi in200-300 ml, 1 h at 37° C.). Peptide-loaded ⁵¹Cr-labeled LCL wereincubated with CD8⁺ T cells at effector-target ration between 5:1 and1.25:1. Cytotoxicity was recorded as percentage ⁵¹Cr released intoculture supernatants.

[0172] SCCE Peptide 5-13

[0173] SCCE peptide 5-13 (SEQ ID No. 33) is an HLA A2.1-binding peptide,as revealed by upregulation of A2.1 expression in T2 cells (data notshown). CD8⁺ CTL specific for SCCE 5-13 killed peptide-loaded autologousLCL, but did not kill control, peptide-free LCL. Heterologous HLAA2.1-expressing peptide-loaded LCL were efficiently killed, but targetslacking HLA A2.1 were not killed (FIG. 20).

[0174] SCCE peptide 123-131

[0175] SCCE peptide 123-131 (SEQ ID No. 32) is also an HLA A2.1-bindingpeptide, as revealed by upregulation of A2.1 expression in T2 cells(data not shown). CD8⁺ CTL specific for SCCE 123-131 killedpeptide-loaded autologous LCL, but did not kill control, peptide-freeLCL. Heterologous HLA A2. 1-expressing peptide-loaded LCL wereefficiently killed, but targets lacking HLA A2.1 were not killed (FIG.21). Natural killer-sensitive K562 cells were not lysed. Cytotoxicityagainst SCCE 123-131 loaded LCL could be blocked with MAb specific for anon-polymorphic HLA class I determinant, confirming that lysis was HLAclass I-restricted.

EXAMPLE 18

[0176] CD8⁺ CTL Specific for SCCE Peptide 123-131 Recognize EndogenouslyExpressed SCCE Tumor Antigen

[0177] To determine whether peptide-specific CD8⁺ CTL are capable ofrecognizing targets that process and present endogenously expressed SCCEtumor antigens, recombinant adenoviruses expressing hepsin and SCCE,both in conjunction with green fluorescent protein (GFP) as a means ofdirectly monitoring expression levels by flow cytometric techniques wereconstructed. It was found that CD8⁺ CTL specific for SCCE 123-131recognize and kill autologous targets infected with recombinantadenoviruses expressing the full-length SCCE antigen (Ad-GFP/SCCE) butdid not recognize targets infected with Ad-GFP/hepsin (FIG. 22). Theseresults show that the SCCE 123-131 peptide is a naturally processed andpresented CTL epitope for SCCE-specific CD8⁺ CTL.

[0178] CD8⁺ CTL specific for SCCE 123-131 were tested for their abilityto lyse HLA-A*0201-matched, SCCE-expressing CaOV-3 ovarian tumor cells.Peptide-specific CTL efficiently lysed peptide-pulsed LCL. The CTL alsolysed CaOV-3 tumor cells (FIG. 23). Thus, SCCE 123-131 is a CTL epitopethat is processed and presented by ovarian tumor cells. Tumor cell lysiswas partially blocked by addition of monoclonal antibody specific forHLA Class I (W6/32) and HLA-A*0201 (BB7.2), indicating that tumor lysiswas HLA Class I- and HLA-A*0201-restricted. Control, unpulsed LCL andNK-sensitive K562 cells were not lysed. These results indicate thatovarian tumor recognition and lysis was an antigen-specific event.

EXAMPLE 19

[0179] SCCE Peptide Expressing Both CD8⁺ CTL And CD4⁺ Helper T CellEpitopes The above results show that dendritic cells loaded with SCCEpeptide 123-131 can stimulate peptide-specific HLA A2-restricted CD8⁺cytotoxic T cells that kill SCCE-expressing, HLA A2-matched ovariantumor cells. The SCCE 123-131 peptide is thus a legitimate target forimmunotherapy of ovarian cancer. However, for optimal dendritic cellimmunotherapy, it is also necessary to stimulate tumor antigen-specifichelper CD4⁺ T cell responses.

[0180] The present example discloses a SCCE peptide that containsepitopes for inducing both CD4⁺ and CD8⁺ T cell responses. The algorithmfor prediction of HLA DR-binding epitopes is built on motifs for DRI,DR4 and DR7. However, DR molecules frequently share commonpeptide-binding motifs, with the result that many peptides showdegenerate binding to multiple DR molecules. Consequently, combinedanalysis of the DRI, DR4 and DR7 motifs has a high probability ofidentifying degenerate epitopes that bind other HLA DR molecules, ormultiple DR-binding clusters (Southwood et al., 1998). Analysis of thecomplete SCCE sequence revealed a region (residues 110- 139) thatpossesses a cluster of candidate HLA DR-binding epitopes as well as theknown HLA 2.1-restricted CTL epitope SCCE 123-131 (see Table 9).

[0181] SCCE 110-139-Loaded Dendritic Cells Stimulate CD4⁺ T CellResponses In Diverse HLA DR Backgrounds

[0182] SCCE 110-139 peptide-loaded dendritic cells were used tostimulate CD4⁺ T cells from two donors. The first donor expressed DR1and DR7, whereas the second donor expressed unrelated DR3 and DR6 ClassII haplotypes. Strong 110-139 peptide-specific CD4⁺ T cell proliferativeresponses were induced from donor 1 (data not shown). Peptide-specificCD4⁺ T cells from donor 1 were also cytotoxic against peptide-pulsedautologous LCL (FIG. 24). It was also found that SCCE 110-139-loadeddendritic cells efficiently stimulated proliferative (data not shown)and cytotoxic CD4⁺ T cell responses from the second donor (FIG. 25).From these results, it was concluded that vaccination with SCCE110-139-loaded dendritic cells is likely to be immunogenic in themajority of individual, and would not be restricted to a limited set ofHLA Class II haplotypes.

[0183] Dendritic Cells Loaded With SCCE 110-139 Efficiently StimulateCD8⁺ CTL Responses

[0184] SCCE 110-139 peptide-loaded dendritic cells also processed andcross-presented epitopes on HLA Class I, efficiently priming a CD8⁺ CTLresponse. Peptide-specific CD8⁺ CTL lysed autologous LCL loaded withSCCE 110-139, showed reduced but significant lysis against A2.1-matchedLCL, and failed to lyse HLA Class 1-mismatched, peptide-loaded LCL (FIG.26). These results provide an important confirmation that CTL epitopesare cross-presented by dendritic cells, and that CD8-CTL response topeptide 110-139 is restricted by multiple Class I molecules, includingA2.1. Intriguingly, the results also suggest that the target LCL arecapable of cross-presenting soluble antigen. Peptide 110-139-specificCD8⁺ CTL did not lyse SCCE 123-131-pulsed target cells, suggesting thatalthough SCCE 123-131 is a naturally processed epitope, it is not amajor component of the CD8⁺ CTL response to the extended 110-139peptide. Rather, the results from FIG. 26 indicate that peptide110-139-specific CD8⁺ CTL recognize other epitopes processed from theSCCE 110-139 sequence and presented by HLA 2.1 and other HLA Class Imolecules. Nevertheless, CD8⁺ CTL specific for SCCE 123-131 can lysepeptide 110-139-loaded target cells (FIG. 27).

[0185] Although peptide 123-131 can be cross-presented by peptide110-139-loaded dendritic cells and recognized by A2.1-restrictedSCCE123-13 1-specific CD8⁺ T cells, the relatively low level of lysisindicates that it is not processed as a major epitope from the extendedSCCE 110-139 sequence. This may explain why CD8⁺ CTL stimulated withpeptide 110-139, although strongly cytotoxic against targets loaded withthe homologous peptide, failed to recognize peptide 123-131-loadedtarget cells. This might appear to be a limitation to the use of 110-139as a vaccine antigen, given that 123-131 is an A2.1-restricted epitopethat is naturally processed and presented by ovarian tumor cells.However, the two peptides can be used independently, i.e. dendriticcells can be loaded with both 110-139 and 123-131 peptides, at least fortreatment of A2.1-positive patients. In addition, these resultsdemonstrate that DC loaded with SCCE 110-139 induce CTL specific forother naturally processed epitopes which are likely to be presented bytumor cells. TABLE 9 Identification of HLA-DR Core Binding MotifsProximal To The HLA A2.1-Binding SCCE Peptide 123-131VNDLMLVKLNSQARLSSMVKKVRLPSRCEP SCCE 110-139 (SEQ ID NO:137) LVKLNSQARDR1 (8.33) (SEQ ID NO:138) VKKVRLPSR DR1 (2.12) (SEQ ID NO:139)LMLVKLNSQ DR4 (8.0) (SEQ ID NO:140) LVKLNSQAR DR4 (2.82) (SEQ ID NO:138)VKKVRLPSR DR4 (3.95) (SEQ ID NO:139) LVKLNSQAR DR7 (12.3) (SEQ IDNO:138)

[0186] The following references were cited herein:

[0187] Egelrud, J invest Dermatol 101:200-204 (1993).

[0188] Hall et al., Mol. Cell. Biol., 3: 854-862 (1983).

[0189] Hansson et al., J BioL Chem., 269:19420-19426 (1994). 5 Nazaruket al., Blood 91:3875-3883 (1998).

[0190] Nijman et al., Eur. J Immunol. 23:1215-1219 (1993).

[0191] Noonan et al., Proc. Natl. Acad. Sci. USA, 87:7160-7164 (1990).

[0192] Parker et al., J. Immunol. 152:163-175 (1994).

[0193] Powell et al., Cancer Research, 53:417-422 (1993).

[0194] Sakanari et al., Biochemistry 86:4863-4867 (1989).

[0195] Santin et al., Obstetrics & Gynecology 96:422-430 (2000).

[0196] Santin et al., Am. J. Obstet. Gynecol. 183:601-609 (2000).

[0197] Shigemasa et al., J Soc Gynecol Invest 4:95-102, (1997).

[0198] SondeIl et al., J Histochem Cytochem 42:459-465 (1994). Southwoodet al., J. Immunol. 160:3363-3373 (1998).

[0199] Torres-Rosedo et al., Proc. Natl. Acad. Sci. USA. 90:7181-7185(1993).

[0200] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

1 140 1 23 DNA Artificial sequence primer_bind 6, 9, 12, 15, 18 senseoligonucleotide primer for amplifying serine proteases, n = Inosine 1tgggtngtna cngcngcnca ytg 23 2 20 DNA Artificial sequence primer_bind 3,6, 9, 12, 15, 18 antisense oligonucleotide primer for amplifying serineproteases, n = Inosine 2 arnarngcna tntcnttncc 20 3 20 DNA Artificialsequence primer_bind 3, 6, 9, 12, 18 antisense oligonucleotide primerfor amplifying serine proteases, n = Inosine 3 arnggnccnc cnswrtcncc 204 24 DNA Artificial sequence primer_bind 6, 15, 18 sense oligonucleotideprimer for amplifying cysteine proteases, n = Inosine 4 carggncartgyggnwsntg ytgg 24 5 20 DNA Artificial sequence primer_bind 3, 6, 15antisense oligonucleotide primer for amplifying cysteine proteases, n =Inosine 5 tanccnccrt trcanccytc 20 6 20 DNA Artificial sequenceprimer_bind 3, 6, 12, 15, 18 sense oligonucleotide primer for amplifyingmetallo-proteases, n = Inosine 6 ccnmgntgyg gnrwnccnga 20 7 17 DNAArtificial sequence primer_bind 6, 9, 11 antisense oligonucleotideprimer for amplifying metallo-proteases, n = Inosine 7 ttrtgnccnanytcrtg 17 8 20 DNA Artificial sequence primer_bind senseoligonucleotide primer specific for hepsin 8 tgtcccgatg gcgagtgttt 20 920 DNA Artificial sequence primer_bind antisense oligonucleotide primerspecific for hepsin 9 cctgttggcc atagtactgc 20 10 20 DNA Artificialsequence primer_bind sense oligonucleotide primer specific for SCCE 10agatgaatga gtacaccgtg 20 11 20 DNA Artificial sequence primer_bindantisense oligonucleotide primer specific for SCCE 11 ccagtaagtccttgtaaacc 20 12 20 DNA Artificial sequence primer_bind senseoligonucleotide primer specific for CompB 12 aagggacacg agagctgtat 20 1320 DNA Artificial sequence primer_bind antisense oligonucleotide primerspecific for CompB 13 aagtggtagt tggaggaagc 20 14 20 DNA Artificialsequence primer_bind sense oligonucleotide primer specific for Cath-L 14attggagaga gaaaggctac 20 15 20 DNA Artificial sequence primer_bindantisense oligonucleotide primer specific for Cath-L 15 cttgggattgtacttacagg 20 16 20 DNA Artificial sequence primer_bind senseoligonucleotide primer specific for PUMP-1 16 cttccaaagt ggtcacctac 2017 20 DNA Artificial sequence primer_bind antisense oligonucleotideprimer specific for PUMP-1 17 ctagactgct accatccgtc 20 18 17 DNAArtificial sequence primer_bind sense oligonucleotide primer specificfor (-tubulin 18 tgcattgaca acgaggc 17 19 17 DNA Artificial sequenceprimer_bind antisense oligonucleotide primer specific for (-tubulin 19ctgtcttgac attgttg 17 20 20 DNA Artificial sequence primer_bind senseoligonucleotide primer specific for Protease M 20 ctgtgatcca ccctgactat20 21 20 DNA Artificial sequence primer_bind antisense oligonucleotideprimer specific for Protease M 21 caggtggatg tatgcacact 20 22 20 DNAArtificial sequence primer_bind sense oligonucleotide primer specificfor TADG-12 22 gcgcactgtg tttatgagat 20 23 20 DNA Artificial sequenceprimer_bind antisense oligonucleotide primer specific for TADG-12 23ctctttggct tgtacttgct 20 24 20 DNA Artificial sequence primer_bind senseoligonucleotide primer specific for TADG-13 24 tgagggacat cattatgcac 2025 20 DNA Artificial sequence primer_bind antisense oligonucleotideprimer specific for TADG-13 25 caagttttcc ccataattgg 20 26 20 DNAArtificial sequence primer_bind sense oligonucleotide primer specificfor TADG-14 26 acagtacgcc tgggagacca 20 27 20 DNA Artificial sequenceprimer_bind antisense oligonucleotide primer specific for TADG-14 27ctgagacggt gcaattctgg 20 28 12 PRT Unknown CHAIN a poly-lysine linkedmultiple Ag peptide derived from SCCE protein sequences 28 Pro Leu GlnIle Leu Leu Leu Ser Leu Ala Leu Glu 5 10 29 12 PRT Unknown CHAIN apoly-lysine linked multiple Ag peptide derived from SCCE proteinsequences 29 Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His 5 10 30 969DNA Homo sapiens mat_peptide full length cDNA of SCCE 30 ttgagggttttgtgtttctt tatttgtttt ggttttaggt ctttaccaat 50 ttgattggtt tatcaacagggcatgaggtt taaatatatc tttgaggaaa 100 ggtaaagtca aatttgactt cataggtcatcggcgtcctc actcctgtgc 150 attttctgtt ggaagcacac agttaattaa ctcagtgtggcgttagcgat 200 gctttttcat ggtgtcattt atccacttgg tgaacttgca cacttgagtg250 tagactcctg ggtcattggg ttggccgcaa gggaaagttc cccaggacac 300cagaccttgc agggtacctc tgcacaccaa cggtccccct gagtcaccat 350 tgcaggcgtttttcttggag tcggggatgc cagcgcacag catggaattt 400 tccagtaagt ccttgtaaaccttcgtgcag tcctgggggg agatgagctt 450 gacatccacg cacatgaggt cagagggaaaggtcacatct gggctcgtgg 500 tagtgcccca gccggagaca gtacaggtgg ttccagggggttcgcagcgg 550 gagggcagcc tgactttctt caccatggat gacagcctgg cctggctatt600 gagcttcacg agcatgaggt cattaacatg ggtctgtgtg gagtagccgg 650ggtggcggaa tgacttcgag gccttgatcc tctgagctct cctgtcgccc 700 agcgtatcactgcccaggtg cacggtgtac tcattcatct tgcagtgggc 750 ggcagtgagc acccagcgctcattgaccag gacgcctccg cagtggagct 800 gattgccact gagcagggcc acctgccatgggtgggagcc tcttgcacat 850 ggggcgccat caataatctt gtcaccctgg gcttcttctcctgcagtttc 900 caaggctaag gatagcagta ggatctgcag gggcaggaga agggatcttg950 ccatggagcc cggaaatcc 969 31 9 PRT Homo sapiens CHAIN Residues 72-80of the SCCE protein 31 Lys Met Asn Glu Tyr Thr Val His Leu 5 32 9 PRTHomo sapiens CHAIN Residues 123-131 of the SCCE protein 32 Arg Leu SerSer Met Val Lys Lys Val 5 33 9 PRT Homo sapiens CHAIN Residues 5-13 ofthe SCCE protein 33 Leu Leu Leu Pro Leu Gln Ile Leu Leu 5 34 9 PRT Homosapiens CHAIN Residues 58-66 of the SCCE protein 34 Val Leu Val Asn GluArg Trp Val Leu 5 35 9 PRT Homo sapiens CHAIN Residues 6-14 of the SCCEprotein 35 Leu Leu Pro Leu Gln Ile Leu Leu Leu 5 36 9 PRT Homo sapiensCHAIN Residues 4-12 of the SCCE protein 36 Ser Leu Leu Leu Pro Leu GlnIle Leu 5 37 9 PRT Homo sapiens CHAIN Residues 52-60 of the SCCE protein37 Gln Leu His Cys Gly Gly Val Leu Val 5 38 9 PRT Homo sapiens CHAINResidues 12-20 of the SCCE protein 38 Leu Leu Leu Ser Leu Ala Leu GluThr 5 39 9 PRT Homo sapiens CHAIN Residues 163-171 of the SCCE protein39 Leu Met Cys Val Asp Val Lys Leu Ile 5 40 9 PRT Homo sapiens CHAINResidues 57-65 of the SCCE protein 40 Gly Val Leu Val Asn Glu Arg TrpVal 5 41 9 PRT Homo sapiens CHAIN Residues 237-245 of the SCCE protein41 Gln Val Cys Lys Phe Thr Lys Trp Ile 5 42 9 PRT Homo sapiens CHAINResidues 169-177 of the SCCE protein 42 Lys Leu Ile Ser Pro Gln Asp CysThr 5 43 9 PRT Homo sapiens CHAIN Residues 10-18 of the SCCE protein 43Gln Ile Leu Leu Leu Ser Leu Ala Leu 5 44 9 PRT Homo sapiens CHAINResidues 29-37 of the SCCE protein 44 Lys Ile Ile Asp Gly Ala Pro CysAla 5 45 9 PRT Homo sapiens CHAIN Residues 215-223 of the SCCE protein45 Leu Gln Gly Leu Val Ser Trp Gly Thr 5 46 9 PRT Homo sapiens CHAINResidues 13-21 of the SCCE protein 46 Leu Leu Ser Leu Ala Leu Glu ThrAla 5 47 9 PRT Homo sapiens CHAIN Residues 114-122 of the SCCE protein47 Met Leu Val Lys Leu Asn Ser Gln Ala 5 48 9 PRT Homo sapiens CHAINResidues 47-55 of the SCCE protein 48 Leu Leu Ser Gly Asn Gln Leu HisCys 5 49 9 PRT Homo sapiens CHAIN Residues 65-73 of the SCCE protein 49Val Leu Thr Ala Ala His Cys Lys Met 5 50 9 PRT Homo sapiens CHAINResidues 59-67 of the SCCE protein 50 Leu Val Asn Glu Arg Trp Val LeuThr 5 51 9 PRT Homo sapiens CHAIN Residues 51-59 of the SCCE protein 51Asn Gln Leu His Cys Gly Gly Val Leu 5 52 9 PRT Homo sapiens CHAINResidues 77-85 of the SCCE protein 52 Thr Val His Leu Gly Ser Asp ThrLeu 5 53 9 PRT Homo sapiens CHAIN Residues 45-53 of the SCCE protein 53Val Ala Leu Leu Ser Gly Asn Gln Leu 5 54 9 PRT Homo sapiens CHAINResidues 162-170 of the SCCE protein 54 Asp Leu Met Cys Val Asp Val LysLeu 5 55 9 PRT Homo sapiens CHAIN Residues 218-226 of the SCCE protein55 Leu Val Ser Trp Gly Thr Phe Pro Cys 5 56 9 PRT Homo sapiens CHAINResidues 145-153 of the SCCE protein 56 Thr Val Ser Gly Trp Gly Thr ThrThr 5 57 9 PRT Homo sapiens CHAIN Residues 136-144 of the SCCE protein57 Arg Cys Glu Pro Pro Gly Thr Thr Cys 5 58 9 PRT Homo sapiens CHAINResidues 81-89 of the SCCE protein 58 Gly Ser Asp Thr Leu Gly Asp ArgArg 5 59 9 PRT Homo sapiens CHAIN Residues 30-38 of the SCCE protein 59Ile Ile Asp Gly Ala Pro Cys Ala Arg 5 60 9 PRT Homo sapiens CHAINResidues 183-191 of the SCCE protein 60 Leu Leu Glu Asn Ser Met Leu CysAla 5 61 9 PRT Homo sapiens CHAIN Residues 21-29 of the SCCE protein 61Ala Gly Glu Glu Ala Gln Gly Asp Lys 5 62 9 PRT Homo sapiens CHAINResidues 235-243 of the SCCE protein 62 Tyr Thr Gln Val Cys Lys Phe ThrLys 5 63 9 PRT Homo sapiens CHAIN Residues 170-178 of the SCCE protein63 Leu Ile Ser Pro Gln Asp Cys Thr Lys 5 64 9 PRT Homo sapiens CHAINResidues 245-253 of the SCCE protein 64 Ile Asn Asp Thr Met Lys Lys HisArg 5 65 9 PRT Homo sapiens CHAIN Residues 157-165 of the SCCE protein65 Val Thr Phe Pro Ser Asp Leu Met Cys 5 66 9 PRT Homo sapiens CHAINResidues 109-117 of the SCCE protein 66 His Val Asn Asp Leu Met Leu ValLys 5 67 9 PRT Homo sapiens CHAIN Residues 17-25 of the SCCE protein 67Ala Leu Glu Thr Ala Gly Glu Glu Ala 5 68 9 PRT Homo sapiens CHAINResidues 151-159 of the SCCE protein 68 Thr Thr Thr Ser Pro Asp Val ThrPhe 5 69 9 PRT Homo sapiens CHAIN Residues 68-76 of the SCCE protein 69Ala Ala His Cys Lys Met Asn Glu Tyr 5 70 9 PRT Homo sapiens CHAINResidues 173-181 of the SCCE protein 70 Pro Gln Asp Cys Thr Lys Val TyrLys 5 71 9 PRT Homo sapiens CHAIN Residues 204-212 of the SCCE protein71 Asp Ser Gly Gly Pro Leu Val Cys Arg 5 72 9 PRT Homo sapiens CHAINResidues 39-47 of the SCCE protein 72 Gly Ser His Pro Trp Gln Val AlaLeu 5 73 9 PRT Homo sapiens CHAIN Residues 222-230 of the SCCE protein73 Gly Thr Phe Pro Cys Gly Gln Pro Asn 5 74 9 PRT Homo sapiens CHAINResidues 165-173 of the SCCE protein 74 Cys Val Asp Val Lys Leu Ile SerPro 5 75 9 PRT Homo sapiens CHAIN Residues 110-118 of the SCCE protein75 Val Asn Asp Leu Met Leu Val Lys Leu 5 76 9 PRT Homo sapiens CHAINResidues 179-187 of the SCCE protein 76 Val Tyr Lys Asp Leu Leu Glu AsnSer 5 77 9 PRT Homo sapiens CHAIN Residues 105-113 of the SCCE protein77 Ser Thr Gln Thr His Val Asn Asp Leu 5 78 9 PRT Homo sapiens CHAINResidues 234-242 of the SCCE protein 78 Val Tyr Thr Gln Val Cys Lys PheThr 5 79 9 PRT Homo sapiens CHAIN Residues 125-133 of the SCCE protein79 Ser Ser Met Val Lys Lys Val Arg Leu 5 80 9 PRT Homo sapiens CHAINResidues 207-215 of the SCCE protein 80 Gly Pro Leu Val Cys Arg Gly ThrLeu 5 81 9 PRT Homo sapiens CHAIN Residues 51-59 of the SCCE protein 81Asn Gln Leu His Cys Gly Gly Val Leu 5 82 9 PRT Homo sapiens CHAINResidues 175-183 of the SCCE protein 82 Asp Cys Thr Lys Val Tyr Lys AspLeu 5 83 9 PRT Homo sapiens CHAIN Residues 103-111 of the SCCE protein83 Gly Tyr Ser Thr Gln Thr His Val Asn 5 84 9 PRT Homo sapiens CHAINResidues 201-209 of the SCCE protein 84 Cys Asn Gly Asp Ser Gly Gly ProLeu 5 85 9 PRT Homo sapiens CHAIN Residues 210-218 of the SCCE protein85 Val Cys Arg Gly Thr Leu Gln Gly Leu 5 86 9 PRT Homo sapiens CHAINResidues 1-9 of the SCCE protein 86 Met Ala Arg Ser Leu Leu Leu Pro Leu5 87 9 PRT Homo sapiens CHAIN Residues 125-133 of the SCCE protein 87Ser Ser Met Val Lys Lys Val Arg Leu 5 88 9 PRT Homo sapiens CHAINResidues 156-164 of the SCCE protein 88 Asp Val Thr Phe Pro Ser Asp LeuMet 5 89 9 PRT Homo sapiens CHAIN Residues 72-80 of the SCCE protein 89Lys Met Asn Glu Tyr Thr Val His Leu 5 90 9 PRT Homo sapiens CHAINResidues 107-115 of the SCCE protein 90 Gln Thr His Val Asn Asp Leu MetLeu 5 91 9 PRT Homo sapiens CHAIN Residues 176-184 of the SCCE protein91 Cys Thr Lys Val Tyr Lys Asp Leu Leu 5 92 9 PRT Homo sapiens CHAINResidues 138-146 of the SCCE protein 92 phe Pro Pro Gly Thr Thr Cys ThrVal 5 93 9 PRT Homo sapiens CHAIN Residues 70-78 of the SCCE protein 93His Val Lys Met Asn Glu Tyr Thr Val 5 94 9 PRT Homo sapiens CHAINResidues 175-183 of the SCCE protein 94 Asp Cys Thr Lys Val Tyr Lys AspLeu 5 95 9 PRT Homo sapiens CHAIN Residues 119-127 of the SCCE protein95 Asn Ser Gln Ala Arg Leu Ser Ser Met 5 96 9 PRT Homo sapiens CHAINResidues 241-249 of the SCCE protein 96 Phe Thr Lys Trp Ile Asn Asp ThrMet 5 97 9 PRT Homo sapiens CHAIN Residues 90-98 of the SCCE protein 97Ala Gln Arg Ile Lys Ala Ser Lys Ser 5 98 9 PRT Homo sapiens CHAINResidues 238-246 of the SCCE protein 98 Val Cys Lys Phe Thr Lys Trp IleAsn 5 99 9 PRT Homo sapiens CHAIN Residues 91-99 of the SCCE protein 99Gln Arg Ile Lys Ala Ser Lys Ser Phe 5 100 9 PRT Homo sapiens CHAINResidues 62-70 of the SCCE protein 100 Glu Arg Trp Val Leu Thr Ala AlaHis 5 101 9 PRT Homo sapiens CHAIN Residues 211-219 of the SCCE protein101 Cys Arg Gly Thr Leu Gln Gly Leu Val 5 102 9 PRT Homo sapiens CHAINResidues 135-143 of the SCCE protein 102 Ser Arg Cys Glu Pro Pro Gly ThrThr 5 103 9 PRT Homo sapiens CHAIN Residues 37-45 of the SCCE protein103 Ala Arg Gly Ser His Pro Trp Gln Val 5 104 9 PRT Homo sapiens CHAINResidues 227-235 of the SCCE protein 104 Gly Gln Pro Asn Asp Pro Gly ValTyr 5 105 9 PRT Homo sapiens CHAIN Residues 236-244 of the SCCE protein105 Thr Gln Val Cys Lys Phe Thr Lys Trp 5 106 9 PRT Homo sapiens CHAINResidues 88-96 of the SCCE protein 106 Arg Arg Ala Gln Arg Ile Lys AlaSer 5 107 9 PRT Homo sapiens CHAIN Residues 87-95 of the SCCE protein107 Asp Arg Arg Ala Gln Arg Ile Lys Ala 5 108 9 PRT Homo sapiens CHAINResidues 233-241 of the SCCE protein 108 Gly Val Tyr Thr Gln Val Cys LysPhe 5 109 9 PRT Homo sapiens CHAIN Residues 72-80 of the SCCE protein109 Lys Met Asn Glu Tyr Thr Val His Leu 5 110 9 PRT Homo sapiens CHAINResidues 122-130 of the SCCE protein 110 Ala Arg Leu Ser Ser Met Val LysLys 5 111 9 PRT Homo sapiens CHAIN Residues 120-128 of the SCCE protein111 Ser Gln Ala Arg Leu Ser Ser Met Val 5 112 9 PRT Homo sapiens CHAINResidues 9-17 of the SCCE protein 112 Leu Gln Ile Leu Leu Leu Ser LeuAla 5 113 9 PRT Homo sapiens CHAIN Residues 215-223 of the SCCE protein113 Leu Gln Gly Leu Val Ser Trp Gly Thr 5 114 9 PRT Homo sapiens CHAINResidues 131-139 of the SCCE protein 114 Val Arg Leu Pro Ser Arg Cys GluPro 5 115 9 PRT Homo sapiens CHAIN Residues 106-114 of the SCCE protein115 Thr Gln Thr His Val Asn Asp Leu Met 5 116 9 PRT Homo sapiens CHAINResidues 2-10 of the SCCE protein 116 Ala Arg Ser Leu Leu Leu Pro LeuGln 5 117 9 PRT Homo sapiens CHAIN Residues 99-107 of the SCCE protein117 Phe Arg His Pro Gly Tyr Ser Thr Gln 5 118 9 PRT Homo sapiens CHAINResidues 137-145 of the SCCE protein 118 Cys Glu Pro Pro Gly Thr Thr CysThr 5 119 9 PRT Homo sapiens CHAIN Residues 61-69 of the SCCE protein119 Asn Glu Arg Trp Val Leu Thr Ala Ala 5 120 9 PRT Homo sapiens CHAINResidues 172-180 of the SCCE protein 120 Ser Pro Gln Asp Cys Thr Lys ValTyr 5 121 9 PRT Homo sapiens CHAIN Residues 23-31 of the SCCE protein121 Glu Glu Ala Gln Gly Asp Lys Ile Ile 5 122 9 PRT Homo sapiens CHAINResidues 74-82 of the SCCE protein 122 Asn Glu Tyr Thr Val His Leu GlySer 5 123 9 PRT Homo sapiens CHAIN Residues 22-30 of the SCCE protein123 Gly Glu Glu Ala Gln Gly Asp Lys Ile 5 124 9 PRT Homo sapiens CHAINResidues 216-224 of the SCCE protein 124 Gln Gly Leu Val Ser Trp Gly ThrPhe 5 125 9 PRT Homo sapiens CHAIN Residues 32-40 of the SCCE protein125 Asp Gly Ala Pro Cys Ala Arg Gly Ser 5 126 9 PRT Homo sapiens CHAINResidues 230-238 of the SCCE protein 126 Asn Asp Pro Gly Val Tyr Thr GlnVal 5 127 9 PRT Homo sapiens CHAIN Residues 227-235 of the SCCE protein127 Gly Gln Pro Asn Asp Pro Gly Val Tyr 5 128 9 PRT Homo sapiens CHAINResidues 111-119 of the SCCE protein 128 Asn Asp Leu Met Leu Val Lys LeuAsn 5 129 9 PRT Homo sapiens CHAIN Residues 191-199 of the SCCE protein129 Ala Gly Ile Pro Asp Ser Lys Lys Asn 5 130 9 PRT Homo sapiens CHAINResidues 91-99 of the SCCE protein 130 Gln Arg Ile Lys Ala Ser Lys SerPhe 5 131 9 PRT Homo sapiens CHAIN Residues 236-244 of the SCCE protein131 Thr Gln Val Cys Lys Phe Thr Lys Trp 5 132 9 PRT Homo sapiens CHAINResidues 82-90 of the SCCE protein 132 Ser Asp Thr Leu Gly Asp Arg ArgAla 5 133 9 PRT Homo sapiens CHAIN Residues 151-159 of the SCCE protein133 Thr Thr Thr Ser Pro Asp Val Thr Phe 5 134 9 PRT Homo sapiens CHAINResidues 181-189 of the SCCE protein 134 Lys Asp Leu Leu Glu Asn Ser MetLeu 5 135 9 PRT Homo sapiens CHAIN Residues 213-221 of the SCCE protein135 Gly Thr Leu Gln Gly Leu Val Ser Trp 5 136 9 PRT Homo sapiens CHAINResidues 141-149 of the SCCE protein 136 Gly Thr Thr Cys Thr Val Ser GlyTrp 5 137 30 PRT Homo sapiens CHAIN Residues 110-139 of the SCCE protein137 Val Asn Asp Leu Met Leu Val Lys Leu Asn Ser Gln Ala Arg Leu 5 10 15Ser Ser Met Val Lys Lys Val Arg Leu Pro Ser Arg Cys Glu Pro 20 25 30 1389 PRT Homo sapiens CHAIN core binding motifs of HLA DR1, DR4 and DR7 138Leu Val Lys Leu Asn Ser Gln Ala Arg 5 139 9 PRT Homo sapiens CHAIN corebinding motifs of HLA DR1 and DR4 139 Val Lys Lys Val Arg Leu Pro SerArg 5 140 9 PRT Homo sapiens CHAIN core binding motifs of HLA DR4 140Leu Met Leu Val Lys Leu Asn Ser Gln 5

What is claimed is:
 1. A method of vaccinating an individual againststratum corneum chymotrytic enzyme (SCCE), comprising the step of:inoculating an individual with a SCCE peptide, wherein said SCCE peptideelicits an immune response in said individual, thereby vaccinating saidindividual against SCCE.
 2. The method of claim 1, wherein said SCCEpeptide is expressed in peptide-loaded dendritic cells or is expressedfrom an expression vector.
 3. The method of claim 1, wherein saidindividual has cancer, is suspected of having cancer or is at risk ofgetting cancer, wherein said cancer is selected from the groupconsisting of ovarian cancer, lung cancer, prostate cancer and coloncancer.
 4. The method of claim 1, wherein the length of said SCCEpeptide is from about 9-residue long to about 30-residue long.
 5. Themethod of claim 4, wherein said peptide is selected from the groupconsisting of SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and
 137. 6.A method of producing immune-activated cells directed toward stratumcorneum chymotrytic enzyme (SCCE), comprising the steps of: exposingimmune cells to a SCCE protein or fragment thereof, wherein saidexposure to said SCCE protein or fragment thereof activates said immunecells, thereby producing immune-activated cells directed toward stratumcorneum chymotrytic enzyme.
 7. The method of claim 6, wherein saidimmune cells are selected from the group consisting of B cells, T cellsand dendritric cells.
 8. The method of claim 6, wherein the length ofsaid stratum corneum chymotrytic enzyme fragment is from about 9-residuelong to about 30-residue long.
 9. The method of claim 8, wherein said9-residue fragment is selected from the group consisting of SEQ ID Nos.31, 32, 33, 34, 35, 36, 80, 86, 99 and
 137. 10. The method of claim 7,wherein said dendritic cells are isolated from an individual prior tosaid exposure, wherein said activated dendritic cells are reintroducedinto said individual subsequent to said exposure.
 11. The method ofclaim 10, wherein said individual has a cancer, is suspected of having acancer or is at risk of getting a cancer, wherein said cancer isselected from the group consisting of ovarian cancer, lung cancer,prostate cancer and colon cancer.
 12. A method of immunotherapy targetedtoward stratum corneum chymotrytic enzyme (SCCE) in an individual,comprising the steps of: a) isolating dendritic cells from saidindividual; b) expressing a SCCE protein or fragment thereof in saiddendritic cells; and c) transferring said dendritic cells back to saidindividual, wherein said dendritic cells would activate SCCE-specificimmune responses in said individual, thereby generating immunotherapytargeted toward SCCE in said individual.
 13. The method of claim 12,wherein said individual has cancer, is suspected of having cancer or isat risk of getting cancer, wherein said cancer is selected from thegroup consisting of ovarian cancer, lung cancer, prostate cancer andcolon cancer.
 14. The method of claim 12, wherein said expression ofSCCE in said dendritic cells is obtained by a mean selected from thegroup consisting of transfection, transduction and loading saiddendritic cells with a SCCE protein or fragment thereof.
 15. The methodof claim 12, wherein the length of said SCCE fragment is from about9-residue long to about 30-residue long.
 16. The method of claim 15,wherein said fragment is selected from the group consisting of SEQ IDNos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and
 137. 17. A method ofimmunotherapy targeted toward stratum corneum chymotrytic enzyme (SCCE)in an individual, comprising the steps of: a) isolating dendritic cellsfrom said individual; b) expressing a SCCE protein or fragment thereofin said dendritic cells; c) exposing immune cells comprising T cellsisolated from said individual to said dendritic cells, wherein saiddendritic cells would generate SCCE-specific T cells from said immunecells; and d) transferring said immune cells back to said individual,wherein said immune cells would activate SCCE-specific immune responsesin said individual, thereby generating immunotherapy targeted towardSCCE in said individual.
 18. The method of claim 17, wherein saidindividual has cancer, is suspected of having cancer or is at risk ofgetting cancer, wherein said cancer is selected from the groupconsisting of ovarian cancer, lung cancer, prostate cancer and coloncancer.
 19. The method of claim 17, wherein said expression of SCCE insaid dendritic cells is obtained by a mean selected from the groupconsisting of transfection, transduction and loading said dendriticcells with a SCCE protein or fragment thereof.
 20. The method of claim17, wherein the length of said hepsin fragment is from about 9-residuelong to about 30-residue long.
 21. The method of claim 20, wherein saidfragment is selected from the group consisting of SEQ ID Nos. 31, 32,33, 34, 35, 36, 80, 86, 99 and
 137. 22. A method of monitoring theefficacy of vaccinating an individual with stratum corneum chymotryticenzyme (SCCE) or SCCE peptide, said method comprises the steps of:vaccinating said individual with said SCCE or SCCE peptide; isolating Tcells from said individual; and measuring immune responses induced bysaid SCCE or SCCE peptide, wherein an increased level of immuneresponses compared to those exhibited by cells from normal individualindicates that said individual has been vaccinated by said SCCE or SCCEpeptide.
 23. The method of claim 22, wherein said immune response isselected from the group consisting of T cell proliferation induced bysaid SCCE or SCCE peptide, frequency of cytokine-secreting T cellsspecific to said SCCE or SCCE peptide and frequency of T cellsexpressing T cell receptor specific to said SCCE or SCCE peptide. 24.The method of claim 22, wherein said SCCE peptide is selected from thegroup consisting of SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and137.
 25. A method of inhibiting expression of endogenous SCCE in a cell,comprising the step of: introducing into said cell a vector comprising asequence complementary to SEQ ID No.30, wherein expression of saidvector in said cell produces SCCE antisense RNA that hybridizes toendogenous SCCE mRNA, thereby inhibiting expression of endogenous SCCEin said cell.
 26. A method of inhibiting stratum corneum chymotryticenzyme protein in a cell, comprising the step of: introducing into saidcell an antibody specific for a stratum corneum chymotrytic enzyme(SCCE) protein or a fragment thereof, wherein binding of said antibodyto said stratum corneum chymotrytic enzyme protein inhibits said stratumcorneum chymotrytic enzyme protein in said cell.
 27. The method of claim26, wherein said SCCE protein fragment is selected from the groupconsisting of SEQ ID Nos. 31, 32, 33, 34, 35, 36, 80, 86, 99 and 137.28. A method of targeted therapy to an individual, comprising the stepof: administering a compound to an individual, wherein said compound hasa therapeutic moiety and a targeting moiety specific for stratum corneumchymotrytic enzyme.
 29. The method of claim 28, wherein said targetingmoiety is selected from the group consisting of an antibody specific forstratum corneum chymotrytic enzyme and a ligand or ligand binding domainthat binds stratum corneum chymotrytic enzyme.
 30. The method of claim28, wherein said therapeutic moiety is selected from the groupconsisting of a radioisotope, a toxin, a chemotherapeutic agent, animmune stimulant and a cytotoxic agent.
 31. The method of claim 28,wherein said individual suffers from a disease selected from the groupconsisting of ovarian cancer, lung cancer, prostate cancer, and coloncancer.
 32. An immunogenic composition, comprising an immunogenicfragment of a stratum corneum chymotrytic enzyme protein and anappropriate adjuvant.
 33. The immunogenic composition of claim 32,wherein the length of said stratum corneum chymotrytic enzyme fragmentis from about 9-residue long to about 30-residue long.
 34. Theimmunogenic composition of claim 33, wherein said 9-residue fragment isselected from the group consisting of SEQ ID Nos. 31, 32, 33, 34, 35,36, 80, 86, 99 and
 137. 35. An oligonucleotide having a sequencecomplementary to SEQ ID No.30.
 36. A composition comprising theoligonucleotide of claim 35 and a physiologically acceptable carrier.37. A method of treating a neoplastic state in an individual in need ofsuch treatment, comprising the step of: administering to said individualan effective dose of the oligonucleotide of claim
 35. 38. The method ofclaim 37, wherein said neoplastic state is selected from the groupconsisting of ovarian cancer, breast cancer, lung cancer, colon cancer,prostate cancer and other cancers in which stratum corneum chymotryticenzyme is overexpressed.
 39. A method of screening for compounds thatinhibit stratum corneum chymotrytic enzyme activity, comprising thesteps of: contacting a sample comprising stratum corneum chymotryticenzyme protein with a compound; and assaying for stratum corneumchymotrytic enzyme protease activity, wherein a decrease in said stratumcorneum chymotrytic enzyme protease activity in the presence of saidcompound relative to stratum corneum chymotrytic enzyme proteaseactivity in the absence of said compound indicates said compoundinhibits stratum corneum chymotrytic enzyme activity.